Factor X deletion mutants and analogues thereof

ABSTRACT

Factor XΔ analogues are provided, as well as pharmaceutical preparations containing such analogues and methods of preparing such analogues. The factor XΔ analogues have a deletion of the amino acids Arg180 to Arg234 and a modification in the region of the amino acid sequence between Gly173 and Arg179 of the factor X amino acid sequence. Such analogues can include a processing site not normally present in factor X, thus allowing for selective conversion of the analogue to an active form. The analogues and preparations have utility in the treatment of a number of blood coagulation disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is the U.S. national phase of PCT/AT98/00046, filedFeb. 27, 1998, which claims priority to Austrian Application A336/97,filed Feb. 27, 1997.

FIELD OF THE INVENTION

The invention relates to factor XΔ analogues having a deletion of theamino acids from Arg180 to Arg234 and a modification in the region ofthe amino acid sequence between Gly173 and Arg179, to preparationscontaining the factor XΔ analogues or factor Xa analogues according tothe invention, as well as to methods of preparing the factor XΔanalogues according to the invention.

BACKGROUND OF INVENTION

After the blood coagulation process has been initiated, the coagulationcascade continues through sequential activation of various proenzymes(zymogens) in the blood to their active forms, the serine proteases.Among them are, inter alia, factor XII/XIIa, factor XI/XIa, factorIX/IXa, factor.,X/Xa, factor VII/VIIa and prothrombin/thrombin. In theirphysiological state, most of these enzymes are only active if associatedto a membrane surface in a complex. Ca ions are involved in many ofthese processes. The blood coagulation will either follow the intrinsicpathway, wherein all protein components are present in the blood, or theextrinsic pathway, wherein the tissue factor plays a critical role.Finally, the wound will close by thrombin cleaving fibrinogen to fibrin.

The prothrombinase complex is responsible for activating prothrombin tothrombin. Thrombin is an important enzyme which can act as aprocoagulant as well as an anticoagulant. The prothrombinase complex, inwhich, inter alia, factor Va (as cofactor) and factor Xa (as serineprotease) are involved, assembles in a Ca-dependent association at thesurface of phospholipids. It is discussed that factor Xa is thecatalytic component of the prothrombinase complex.

Factor X (Stuart-Prower factor) is a vitamin K-dependent coagulationglycoprotein which can be activated by the intrinsic and the extrinsicblood coagulation cascade. The primary translation product of factor X(pre-pro-FX) has 488 amino acids and is synthesized by the liver orhuman hepatoma cells initially as a single chain 75 kD precursorprotein. In plasma, factor X is largely present as a double chainmolecule (Fair et al., 1984, Blood 64:194-204).

During biosynthesis, after cleavage of the pre-sequence by a signalpeptidase (between Ser23/Leu24) and of the propeptide (betweenArg40/Ala41), the single chain factor X molecule is cleaved byprocessing and removal of the tripeptide Arg180-Lys181-Arg182 to thedouble chain form consisting of the approximately 22 kD light chain andthe approximately 50 kD heavy chain, which are connected via a disulfidebridge (FIG. 2A, Panel 1A). Therefore, factor X circulates in the plasmaas a double chain molecule.

During the blood coagulation process, factor X is converted frominactive zymogen to active protease factor Xa by limited proteolysis,wherein factor X can be activated to factor Xa in either of twomembrane-associated complexes: in the extrinsic factor VIIa-tissuefactor complex or in the intrinsic factor VIIIa-factorIXa-phospholipid-Ca complex, or “tenase complex” (Mertens et al., 1980,Biochem. J. 185:647-658). A proteolytic cleavage between amino acidsArg234/Ile235 results in the release of an activation peptide having alength of 52 amino acids from the N-terminus of the heavy chain and thusto the formation of the active enzyme, factor Xa. The catalytic centerof factor Xa is located on the heavy chain.

Activation via the factor VIIa-TF (extrinsic) complex results in theformation of Factor Xaα (35 kD) and factor Xaβ (31 kD), with apolypeptide of 42 (kD) forming, too, if the factor VIIa,concentration inthe complex is low. Factor Xaα is formed by a cleavage at Arg234/Ile235of the heavy chain and represents the activation of factor X to factorXa. The occurence of factor Xaβ presumably results from an autocatalyticcleavage at Arg469/Gly470 in the C-terminus of the heavy chain of factorXaα and the cleavage of a 4.5 kD peptide. Like factor Xaα, factor Xaβhas catalytic activity. It has been shown, however, that a plasminogenreceptor binding site is formed by the cleavage of factor Xaα to factorXaβ, and that factor Xaβ optionally has fibrinolytic activity or isinvolved in fibrinolysis as a cofactor. The transformation of factor Xaαto factor Xaβ, however, is slower than the formation of thrombin, thuspreventing the initiation of fibrinolysis before a blood clot is formed(Pryzdial et al., 1996, J. Biol. Chem. 271:16614-16620; Pryzdial et al.,1996, J. Biol. Chem. 271:16621-16626).

The 42 kD polypeptide results from processing in the C-terminus of theheavy chain between Arg469/Gly470 without previous processing betweenArg234/Ile235. Like a factor Xaγ fragment formed by proteolysis atLys370, this intermediate has no catalytic activity (Mertens et al.,1980, Biochem. J. 185:647-658; Pryzdial et al., 1996, J. Biol. Chem.271:16614-16620).

Intrinsic factor X activation is catalysed by the factor IXa-factorVIIIa complex. The same processing products are obtained duringactivation, but the factor Xaβ product is obtained in a larger quantitythan other factor X processing products (Jesty et al., 1974, J. Biol.Chem. 249:5614).

In vitro, factor X can, for instance, be activated by Russell's vipervenom (RVV) or trypsin (Bajaj et al., 1973, J. Biol. Chem.248:7729-7741) or by purified physiological activators, such as FVIIa/TFcomplex or factor IXa/factor VIIIa complex (Mertens et al., 1980,Biochem. J. 185:647-658).

Most commercially available factor X products from plasma contain amixture of factor Xaα and factor Xaβ, because after activation of factorX to factor Xa mainly factor Xaα is formed, which is, in turn, cleavedto factor Xaβ in an autocatalytic process.

In order to produce a uniform factor Xa product having high molecularintegrity, EP 0 651 054 suggested to activate factor X with RVV over anextended period of time so that the resulting final productsubstantially contains factor Xaβ. The by-products, e.g. factor Xaα, aswell as the protease were subsequently removed by severalchromatographic steps.

cDNA for factor X has been isolated and characterized (Leytus et al.,1984, Proc. Natl. Acad. Sci., U.S.A., 82:3699-3702; Fung et al., 1985,Proc. Natl. Acad. Sci., U.S.A., 82:3591-3595). Human factor X has beenexpressed in vitro in various types of cells, such as human embryonalrenal cells or CHO cells (Rudolph et al., 1997, Prot. Expr. Purif.10:373-378, Wolf et al., 1991, J. Biol. Chem. 266:13726-13730). However,it was found that in the recombinant expression of human factor X, theprocessing at position Arg40/Ala41 is inefficient, as opposed to thesituation in viva, and that different N-termini form at the light chainof factor X (Wolf et al., 1991, J. Biol. Chem. 266:13726-13730).Recombinant factor X (rFX) was activated to rfactor Xa (rFXa) by RVV invitro, or rFXa was expressed directly, with the activation peptide beingdeleted from amino acid 183 to amino acid 234 and replaced by atripeptide in order to allow processing directly to a double chain rFXaform. About 70% of purified rFX was processed to light and heavy chain,while the remaining 300 represented single chain rFX of 75 kD. Directexpression of rFXa did result in the formation of active factor Xa, butalso of inactive intermediates. Furthermore, Wolf et al. (1991, J. Biol.Chem. 266:13726-13730) detected still reduced activity of recombinantfactor X, which they ascribed to the poorer ability of rFX to beactivated by RVV and to the inactive protein and polypeptide populationsof the single chain precursor molecule. In particular, they found highrFXa instability when expressed by recombinant cells, which theyascribed to the high rate of autoproteolysis.

In order to study the function of the C-terminal peptide of factor Xaα,Eby et al. (1992, Blood 80 (suppl. 1): 1214 A) introduced a stop codonat position Gly430 of the factor X sequence. However, they did not finda difference between the rate of activation of factor Xa (Fxaα) withβ-peptide or a deletion mutant without β-peptide (FXaβ).

Factor Xa is an important component of the prothrombinase complex and istherefore under discussion as a primary mediator for quick hemostasis,and thus it seems suitable for the treatment of patients suffering fromblood coagulation disorders, e.g. hemophilia.

Particularly the treatment of hemophilia patients suffering from factorVIII or factor IX deficiency with factor concentrates produced fromplasma is often complicated by the formation of inhibiting antibodiesagainst these factors in long-term therapy. Therefore, a number ofalternatives have been developed to treat hemophiliacs with factorshaving bypass activity. The use of prothrombin complex concentrate,partially activated prothrombinase complex (APPC), factor VIIa or FEIBAhas been suggested. Commercial preparations having factor VIII bypassactivity (FEIBA) are, for instance, FEIBA® or Autoplex®. FEIBA, containscomparable units of factor II, factor VII, factor IX, factor X andFEIBA, small amounts of factor VIII and factor V, and traces ofactivated coagulation factors, such as thrombin and factor Xa or afactor having factor X-like activity (Elsinger, 1982, ActivatedProthrombin Complex Concentrates. Ed. Mariani, Russo, Mandelli, pp.77-87). Elsinger particularly points at the importance of a “factorXa-like” activity in FEIBA. Factor VIII bypass activity was shown byGiles et al (1988, British J. Haematology 9:491-497) for a combinationof purified factor Xa and phospholipids in an animal model.

Therefore, factor X/Xa or factor X/Xa-like proteins, either alone or asa component of a coagulation complex, are in high demand and can be usedin various fields of application in hemostasis therapy.

In vivo as well as in vitro, the half-life of factor Xa is considerablyshorter than the half-life of the zymogen. For instance, factor X can bestored stably in glycerol for 18 months, while factor Xa is stable foronly 5 months under the same conditions (Bajaj et al., 1973, J. Biol.Chem. 248:7729-7741) and shows reduced activity by more than 60% after 8months in glycerol at 4° C. (Teng et al., 1981, Thrombosis Res.22:213-220). The half-life of factor Xa in serum is a mere 30 seconds.

Because factor X is instable, the administration of factor Xpreparations has been suggested (U.S. Pat. No. 4,501,731). If, however,the bleeding is so serious that the patient might die, particularly in ahemophiliac, the administration of factor X is ineffective, becauseowing to the functional “tenase complex” deficiency in the intrinsicpathway of blood coagulation, factor X can not be sufficiently activatedto factor Xa, and activation via the extrinsic pathway is often too slowto show effects quickly. Moreover, hemophiliacs have sufficient amountsof factor X, but its prothrombinase activity is 1000 times less thanthat of factor Xa. In such cases it is necessary to administer activatedfactor Xa directly, optionally in combination with phospholipids, asdescribed in Giles et al. (1988, British J. Haematology 9:491-497) orwith other coagulation factors, e.g. with factor VIII bypass activity.

In the preparation of factor Xa from factor X, activation so far mostlyhas been carried out by non-physiological activators of animal origin,such as RVV or trypsin, and it was necessary to make absolutely surethat the final product is completely free of these proteases. Asmentioned above, when factor X is activated to factor Xa, quite a numberof intermediates, some of them inactive, are formed (Bajaj et al., 1973,J. Bio. Chem. 248:7729-7741; Mertens et al., 1980, Biochem. J.185:647-658). The presence of such intermediates results in reducedspecific activity of the product and may produce intermediates which canfunction as active serine protease antagonists. Therefore,the.preparation of a uniform, pure product having high specific activityaccording to conventional methods requires complex processes ofactivation and chromatographic purification.

SUMMARY OF INVENTION

Thus, the aim of the present invention is to provide a preparationcontaining a polypeptide having factor X/Xa activity which exhibits highstability and can be activated to factor Xa without using any of theusual proteases, particularly those of animal origin, such as, forinstance, RVV or trypsin. Another aim is to provide a pharmaceuticalpreparation having factor VIII bypass activity.

According to the present invention, the aim is reached by providing afactor X analogue having a deletion of the amino acids Arg180 to Arg234of the factor X amino acid sequence and a modification of this factor Xdeletion mutant in the region of the amino acid sequence between Gly173and Arg179. By the deletion of the amino acid sequence from Arg180 toArg234, the tripeptide Arg180 to Arg182 as well as the activationpeptide Ser183 to Arg234 are deleted, and the light and heavy chains offactor X and the amino acids Arg179 and Ile235 are directly fused. Thisfusion sequence, however, does not contain a natural cleavage site for aprotease. By modifying the region of the factor X sequence between aminoacid Gly173 and Arg179 and optionally of Ile235, a factor X deletionmutant according to the present invention is obtained, which has a noveldetection and processing site not occurring at this position in thepolypeptide for a protease which would not usually cleave thepolypeptide at this position. Said modification is, at least, anexchange of at least one amino acid between position Gly173 and Arg179and, optionally of Ile235 of the factor X amino acid sequence. Theposition of amino acids refers to the numbering according to thesequence presented in FIGS. 1A and 1B, starting with Met1 and endingwith Lys488. In order to simplify the nomenclature, the amino acidnumbering given for the complete factor X sequence is adhered to for themodified factor X deletion mutant according to the present invention,but said modified factor X deletion mutant will hereinafter be referredto as factor XΔ analogue.

Said modification can be a substitution of at least one amino acid, oran insertion of a peptide sequence representing a protease recognitionor cleavage site. In the factor XΔ analogue according to the presentinvention, the modification is preferably such that it represents arecognition and cleavage sequence for a protease from the group ofendoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE4, LPC/PC7 (as described in Barr et al., 1991, Cell 66:1-3 or in U.S.Pat. No. 5,460,950), serine proteases, such as factor IIa, factor VIIa,factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or aderivative of these proteases.

Preferably, said modification is selected such that processing by one ofthese proteases leads to a polypeptide corresponding to native factor Xain its biological activity and displaying factor Xa activity. Foroptimal processing, it may be necessary in individual cases to exchangethe amino acid Ile235, too. Preferably, however, the NH₂-terminal aminoacid isoleucine of the heavy chain should still be maintained afteractivation, because isoleucine represents one of those amino acids whichperform an essential function in the formation of the substrate bindingpocket (Watzke. et al., 1995, Molecular Basis of Thrombosis andHemostasis, ed. Katherine High & Harold Roberts). The factor XΔanalogues according to the present invention display a structuraldifference, particularly on the amino acid level, as compared to anative factor X sequence, but after activation their activity iscomparable to that of naturally occurring factor X or factor Xa,respectively.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show the nucleotide and amino acid sequence of factor X(SEQ ID NOS:43 and 44, respectively).

FIGS. 2A and 2B show a schematic representation of the factor XΔanalogues having modified protease cleavage sites (SEQ ID NOS:102-119).

FIG. 3 shows a schematic representation of the expression vectorphAct-rFX.

FIGS. 4A and 4B show a Western blot analysis of rfactor X expressed inCHO cells before and after amplification.

FIGS. 5A and 5B show a Western blot analysis of rfactor X after in vitrocleavage by furin derivatives.

FIG. 6 shows a Western blot analysis of rfactor X molecules expressed infurin containing and furin deficient cells.

FIG. 7 shows a schematic representation of rfactor XΔ analogueconstructs having modified C-termini of the heavy chain (SEQ ID NOS:102,103 and 120-122).

FIG. 8 shows a schematic representation of the N-termini of rfactor Xprocessing products from CHO, CHO/r-furin and furin deficient cells (SEQID NOS:99-104).

FIG. 9 shows a Western blot analysis of rfactor XΔ^(RVTR/I) expressed inCHO cells.

FIGS. 10A and 10B show a Western blot analysis of rfactor XΔ^(RVTR/I)after in vitro activation with furin derivative.

DETAILED DESCRIPTION

The invention exemplary provides a number of factor XΔ analogues havinga deletion and, in addition, a modification between Gly173 and Arg179and optionally of Ile235. Modifications can be at one or more positionsin the region between amino acids Gly173 and Arg179, and optionallyIle235, based on the factor X sequence numbered from Met1 to Lys488according to FIGS. 1A and 1B. Amino acid substitutions can be atpositions Ile235 (R1), Arg179, Glu178 (R2), Leu177 (R3), Thr176 (R4),Gln175 (R5) and Lys174 (R6), with Arg179, however preferably remainingunchanged.

Preferably, the factor XΔ analogues according to the invention contain afactor X sequence with Gly173-R6-R5-R4-R3-R2-Arg179-R1 (SEQ ID NO:45),wherein R1=Ile, Val, Ala, Ser or Thr; R2=Glu, Thr, Pro, Gly, Lys or Arg;R3=Leu, Phe, Lys, Met, Gln, Ser, Val, Arg or Pro; R4=Thr, Asn, Asp, Ile,Ser, Met, Pro, Arg or Lys; R5=Asn, Lys, Ser, Glu, Gln, Ala, His or Arg;and R6=Arg, Asp, Phe, Thr, Leu or Ser.

Preferred embodiments of the factor X analogues according to theinvention are factor X analogues having a modification with

a) R1=Ile, R2=Thr, R3=Leu, R4=Asn and optionally R5=Asn and/or R6=Asp(SEQ ID NOS:46-49), and processed by factor VIIa or factor IXa;

b) R1=Val, R2=Thr, R3=Phe, R4=Asp, and optionally R5=Asn and/or R6=Pheand/or R1=Ile or Val (SEQ ID NOS:50-57) (FIG. 2A, panel A), andprocessed by factor XIa;

c) R1=Ile or Val, R2=Pro, R3=Lys, R4=Ile, and optionally R5=Lys and/orR6=Thr (SEQ ID NOS:58-61) (FIG. 2A, panel C), or

R1=Ile, R2=Thr, R3=Ser, R4=Thr, and optionally R5=Lys and/or R6=Thr (SEQID NOS:62-65) (FIG. 2A, panel I), and processed by factor XIIa;

d) R1=Ile or Val, R2=Thr, R3=Met, R4=Ser, and optionally R5=Ser and/orR6=Leu (SEQ ID NOS:66-69) (FIG. 2A, panel D), and processed bykallikrein;

e) R1=Ile, R2=Gly, R3=Gln, R4=Pro, and optionally R5=Lys and/or R6=Ser(SEQ ID NOS:70-73) (FIG. 2A, panel H), or

R1=Ile, R2=Gly, R3=Glu, R4=Ile (SEQ ID NO:74) (FIG. 2A, panel F), or

R1=Ile, R2=Thr, R3=Lys, R4=Met (SEQ ID NO:75) (FIG. 2A, panel E), andprocessed by factor Xa;

f) R1=Ile, R2=Lys, R3=Arg, R4=Arg, and optionally R5=Glu and/or R6=Leu(SEQ ID NOS:76-79), or

R1=Ile, R2=Thr, R3=Val, R4=Arg, and optionally R5=Ala and/or R6=Leu (SEQID NOS:80-83), or

R1=Ile, R2=Arg, R3=Val, R4=Arg, and optionally R5=Gln and/or R6=Leu (SEQID NOS:84 and 85), or

R1=Ile, R2=Arg, R3=Arg, R4=Arg, and optionally R5=His and/or R6=Leu (SEQID NOS:86-89), or

R1=Ile, R2=Lys, R3=Pro, R4=Arg, and optionally R5=Asn and/or R6=Leu (SEQID NOS:90-93), or

R1=Ile, R2=Lys, R3=Arg, R4=Ile, and optionally R5=Arg and/or R6=Leu (SEQID NO:94-97), or

R1=Ile, R2=Lys, R3=Ser, and R4=Arg (SEQ ID NO:98), or

R1=Ile, R2=Thr, R3=Val, and R4=Arg (SEQ ID NO:99), or

R1=Ile, R2=Lys, R3=Leu, and R4=Arg (SEQ ID NO:100) (all see FIG. 2A,panel G), with the sequences mentioned under f) being processed by adibasic endoprotease, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4,PACE 4, LPC/PC7, or a derivative of one of these proteases.

FIGS. 2A and 2B show a possible selection of modifications and aminoacid exchanges leading to changed protease specificity.

The modifications can be carried out by, for instance, directed in vitromutagenesis or PCR or other methods of genetic engineering known fromthe state of the art which are suitable for specifically changing a DNAsequence for directed exchanges of amino acids.

According to the present invention, the factor XΔ analogue of theinvention is preferably activated to a factor Xa analogue by a proteaseselected from the group of endoproteases, such as kexin/Kex2,furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7, serine proteases, suchas factor IIa, factor VIIa, factor IXa, factor XIIa, factor XIa, factorXa, or kallikrein, or a derivative of these proteases.

The factor XΔ analogues according to the invention are present as singlechain polypeptides in enzymatically inactive form. Active factor Xaanalogues are only obtained by cleavage by a protease to the doublechain form. Thus, the modification allows activation of the inactive,single chain factor XΔ analogue polypeptide to the double chain activeform.

One of the difficulties in the preparation of active factor Xa is itsinstability, because autocatalysis results in the formation of other,inactive intermediates besides factor Xaα and factor Xaβ.

For the preparation of essentially intact, active factor X/Xa and factorX/Xa-like molecules, respectively, it would therefore be desirable toobtain only such proteins as result in stable final products.

It is well known that a preferred cleavage site for the processing offactor Xaα (FXaα) to factor Xaβ (FXaβ) is between Arg469/Gly470. Basedon research by Eby et al. (1992, Blood. Vol. 80, Suppl. 1, 1214), nextto a prominent carboxy-terminal peptide (amino acid residues 476-487) offactor X, another, shorter peptide (amino acid residues 474-477) isfound which is formed by autocatalysis of factor Xaα. In order to focusdirected processing of intact factor X to essentially active factor Xawithout obtaining inactive processing intermediates, the factor XΔanalogues of the invention optionally have further modifications.

Therefore, according to a particular embodiment, the factor XΔ analoguesaccording to the invention have one further modification in theC-terminal region of the factor X amino acid sequence.

According to one embodiment, a factor XΔ analogue as described above hasan intact β-peptide (FXha). The factor XΔ analogues according to theinvention particularly have a modification in the region of theC-terminal β-peptide cleavage site which prevents cleavage of theβ-peptide from factor X after activation of factor XΔ to factor Xaanalogue. Thus a factor Xa molecule is obtained which can be isolated upto 100% as intact factor Xaα molecule.

Said modification can be a mutation, deletion or insertion in the regionof the factor X amino acid sequence between amino acid position Arg469and Ser476 and optionally of Lys370. However, an amino acid substitutionis preferred which prevents the polypeptide from folding as aconsequence of the amino acid exchange, which would influence thestructure and thus possibly the function and activity of the protein.

According to one embodiment, the factor XΔ analogues of the inventionhave one of the amino acids at position Arg469 and/or Gly470 exchanged,with Arg469 being preferably exchanged for Lys, His or Ile, and Gly470being preferably exchanged for Ser, Ala, Val or Thr.

Besides a mutation at position Arg469 and/or Gly470, the factor XΔanalogues according to the invention can have a further mutation atposition Lys370 and/or Lys475 and/or Ser476. Amino acid substitution atthis (these) position(s) prevents processing of factor Xaα analogue tofactor Xaβ analogue or C-terminal truncated factor Xa analogues,respectively, because the natural occurring sequence(s) is (are)modified such that an occasional autocatalytic cleavage of acarboxy-terminal peptide becomes impossible.

According to a different embodiment, the factor X analogues of theinvention have deleted carboxy terminal β-peptide (FXΔβ). Such a factorX analogue can be prepared by expressing a cDNA coding for factor XΔanalogue in a recombinant expression system, cloning only thosesequences that code for the amino acids Met1 to Arg179/Ile235 to Arg469.

According to a further embodiment, the factor XΔ analogues according tothe invention have a translation stop signal in the C-terminal region ofthe factor X sequence. This translation stop signal is preferablylocated at a position following a C-terminal amino acid formed afternatural processing. Therefore, the translation stop signal is preferablyat the position of amino acid 470 of the factor X sequence, so that theterminal Arg469 of factor XΔβ is retained. For this purpose, the codonGGC encoding the amino acid Gly470 is substituted by TAA, TAG or TGA.

Another aspect of the present invention relates to factor XΔ analogueswhich are activated to factor Xa analogues by treatment with anappropriate protease in vitro, i.e. the activated factor XΔ analogues.Depending on the factor XΔ analogue used and activated, a factor XaΔanalogue is obtained which, at the C-terminal end of the light chain,has corresponding amino acid modifications, as compared to the naturalfactor Xa sequence. According to the invention, these modifications are,however, selected in such a way as not to negatively affect thebiological activity.

If such a factor X analogue additionally has a translation stop signalin the C-terminal region of the β-peptide, modified factor Xaβ moleculesare obtained. If, however, a factor X analogue is employed which hasmodification(s) within the β-peptide sequence resulting in the β-peptidenot being cleaved off, a factor Xaα analogue with an amino acid exchangein the C-terminus of the molecule is obtained.

The factor XΔ analogues according to the invention only havemodifications which change the specificity for the ability to beactivated and do not significantly influence the activity. Therefore, inany case, biologically and functionally active factor Xa molecules orfactor Xa analogues, respectively, are obtained.

In vitro activation can be effected by a protease selected from thegroup of endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2,PC4, PACE 4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa,factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or aderivative of these proteases. It is within the scope of the presentinvention to use any protease, except RVV or trypsin, as long as it isapt to process the factor XΔ analogue according to the invention tofactor Xa analogue.

Although Wolf et al. (1991, J. Biol. Chem. 266:13726-137309), forinstance, have assumed that an endopeptidase, such as Kex2, furin orPACE, is involved in the processing of the factor Xa deletion mutantdescribed by this group, they do not give a hint as to the influence ofone of these proteases on the processing of factor X. Similarly, U.S.Pat. No. 5,660,950 describes the recombinant preparation of PACE and theuse of the protease to improve processing of vitamin K dependentproteins. In a long list of blood factors, factor X is mentioned amongothers, but no data are provided to verify this statement.

The present invention demonstrates unambiguously for the first time thata protease necessary for the maturing process of factor X is a dibasicendoprotease, particularly endogenic furin. In vivo, the endoproteasemainly mediates the cleavage of the single chain factor X molecule tothe mature form consisting of heavy and light chain. In vitro, it alsomediates the cleavage of the factor X propeptide sequence (Example 2).

According to a particular embodiment, a factor XΔ analogue is providedwhich is preferably present in purified form as a single chain molecule.Factor XΔ analogues having in the modified region a cleavage site for aprotease not present in recombinant cells are obtained after expressionas single chain molecules. The single chain factor XΔ molecule isparticularly characterized by high stability and molecular integrity. Sofar, a single chain, inactive factor XΔ molecule could not be isolatedin purified form, because in recombinant cells it is processed to factorXa and a number of other, also inactive, intermediates (Wolf et al.,1991, J. Biol. Chem. 266:13726-13730). The isolated single chain factorXΔ analogue can be activated by specific processing directly to thedouble chain factor Xa analogue form. This can be effected by bringing asingle chain factor XΔ molecule isolated from a recombinant cell intocontact with a protease cleaving the activation site present in thefactor XΔ analogue. If, for example, a factor XΔ analogue having a furinactivation site is expressed in a furin deficient cell, it can beisolated as a single chain factor XΔ analogue and processed to anactive, double chain factor XΔa analogue by bringing it into contactwith a dibasic protease, such as furin/PACE or Kex2. Factor XΔ analogueshaving a processing site for serine protease or kallikrein can also beisolated as single chain molecules in furin expressing cells and thenprocessed with the serine protease to active factor Xa analogues.

Due to the selective and directed processing reaction, a factor Xaanalogue thus obtained has high stability and structural integrity and,in particular, is free of inactive factor X/Xa analogue intermediatesand autoproteolytic decomposition products.

According to the present invention, the factor XΔ analogue of theinvention is provided in the form of a factor XΔa having intactβ-peptide as well as in the form of a factor XΔ analogue having adeletion of the β-peptide.

Another aspect of the present invention relates to recombinant DNAencoding the factor XΔ analogues of the invention. Said recombinant DNAresults after expression in a factor XΔ analogue with an amino acidsequence corresponding to human factor X except for a deletion of aminoacids from Arg180to Arg234 and a modification allowing processing andactivation to active factor Xa analogues having both intact as well asdeleted β-peptide.

A further aspect of the invention relates to a preparation containing apurified factor XΔ analogue having a deletion of amino acids fromArg180to Arg234 and a modification of amino acids in the region betweenGly173 and Arg179 and optionally of Ile235. Said modification leads to anovel recognition or cleavage site not naturally located at thisposition in the polypeptide for a protease which usually does notprocess the polypeptide at this position. Said preparation can be apurified preparation containing single chain factor XΔ analogue, thepolypeptides being obtained from a cell culture system either afterisolation from the cell culture supernatant or from a cell cultureextract. A recombinant factor XΔ analogue prepurified from a cellculture system can be further purified by methods known from the priorart. Chromatographic methods are particularly useful for this purpose,such as gel filtration, ion exchange or affinity chromatography.

According to one embodiment, the preparation according to the inventioncontains the factor XΔ analogue as a single chain molecule inenzymatically inactive form, with the factor XΔ analogue having a purityof at least 80%, preferably at least 90%, particularly preferably atleast 95%, and the purified preparations containing no inactive,proteolytic intermediates of factor X/Xa analogues.

According to a particular aspect, the preparation contains single chainfactor XΔ analogue having a modification allowing activation to factorXa analogues by one of the proteases selected from the group of dibasicendoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa, factorIXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or a derivativeof these proteases. The activation is effected by bringing the factor XΔanalogue into contact with the appropriate protease, which cleaves atthe modified sequence, whereby a factor Xa analogue is obtained.

In the preparation according to the invention, the factor XΔ analoguecan be present either as factor XΔα (FXΔα) having intact β-peptide, oras factor XΔβ having a deletion of the β-peptide or other C-terminaldeletions.

According to a further embodiment, the preparation according to thepresent invention contains the factor XΔ analogue preferably as a singlechain molecule in isolated form. For this purpose, factor XΔ analogue isobtained, for instance, by recombinant preparation, as a single chainmolecule having one modification allowing activation to factor Xaanalogue in vitro. The activation of factor XΔ analogue to factor Xaanalogue can be effected by bringing factor X analogue into contact witha protease selected from the group of dibasic endoproteases, such askexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7, serineproteases, such as factor IIa, factor VIIa, factor IXa, factor XIIa,factor XIa, factor Xa, or kallikrein, or a derivative of theseproteases. The protease can be immobilized on a carrier.

The preparation according to the invention can serve as a startingmaterial for the production and recovery of factor Xa analogues. Forlarge-scale production, the preparation containing single chain factorXΔ analogue is brought into contact with an optionally immobilizedprotease under conditions allowing optimal activation of factor XΔanalogue to factor Xa analogue, and factor Xa analogues are obtained.The factor Xa analogue thus recovered can subsequently be purified bygenerally known methods and formulated to a pharmaceutical compositionhaving factor Xa activity.

According to a further aspect of the present invention, a preparation isprovided containing a factor Xa analogue having high stability andstructural integrity, which is particularly free of inactive factor X/Xaanalogue intermediates and autoproteolytic decomposition products. It isobtainable by activating a factor XΔ analogue of the above-defined typeand preparing a corresponding preparation.

According to a particular embodiment, the preparation containing thepurified, single chain or double chain factor XΔ analogue contains aphysiologically acceptable carrier and is optionally formulated as apharmaceutical preparation. The formulation can be effected according toa method common per se, and it can be mixed with a buffer containingsalts, such as NaCl, CaCl₂, and amino acids, such as glycin and/orlysin, at a pH in the range of 6 to 8 and formulated as a pharmaceuticalpreparation. The purified preparation containing factor X analogue canbe provided as a storable product, as a ready-made solution,lyophilisate or deep frozen until final use. Preferably, the preparationis stored in lyophilized form and dissolved with an appropriatereconstitution solution to an optically clear solution.

However, the preparation according to the present invention can also beprovided as a liquid preparation or in the form of deep frozen liquid.

The preparation according to the invention is particularly stable, i.e.it can be left standing in dissolved form over an extended period oftime before application. It has appeared that the preparation accordingto the invention suffers no loss in activity for several hours up todays.

The preparation according to the invention can be provided in anappropriate device, preferably an application device, in combinationwith a protease selected from the group of endoproteases, such askexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7, serineproteases, such as factor IIa, factor VIIa, factor IXa, factor XIIa,factor XIa, factor Xa, or kallikrein, or a derivative of theseproteases.

The preparation according to the invention containing a factor XΔanalogue in combination with a protease able to activate the factor XΔanalogue to factor Xa analogue can be provided as a combinationpreparation consisting of a vessel containing a protease immobilized ona carrier, optionally in the form of a small column or a syringe chargedwith an immobilized protease, and a vessel containing the pharmaceuticalpreparation with factor XΔ analogue. For activation of the factor XΔanalogue, the solution containing the factor XΔ analogue is pressed overthe immobilized protease, for instance. During storage of thepreparation, the solution containing factor XΔ analogue is preferablykept apart from the immobilized protease. The preparation according tothe invention can be present in the same vessel as the protease, withthe components, however, being separated in space by an impermeableseparation wall which can be easily removed to use the product. Thesolutions can also be stored in individual vessels and brought intocontact only shortly before application.

In a particular embodiment, the protease used for activation is a serineprotease naturally involved in blood coagulation, such as factor XIIa,which need not be separated from the activated factor Xa analogue beforeapplication but can be applied together with it.

Factor XΔ analogue can be activated to factor Xa analogue shortly beforedirect use, i.e. before application to the patient. The activation canbe effected by bringing it into contact with an immobilized protease orby mixing solutions containing a protease on the one hand and factor XΔanalogue on the other. Thus, it is possible to keep the two componentsin solution separately and to mix them by means of an appropriate devicewherein the components get into contact with each other while passingthrough, and thus to activate factor XΔ analogue to factor Xa analogue.The patient will be administered a mixture of factor Xa and anotherserine protease which has effected the activation. Particular care hasto be taken as regards the dosage, because endogenous factor X isactivated by the additional administration of a serine protease, whichmight result in shorter clotting time.

According to a preferred embodiment, the pharmaceutical preparation isprovided in an appropriate device, preferably an application device,either in frozen liquid or in lyophilized form. An appropriateapplication device can be a double compartment syringe as described inAT 366 916 or AT 382 783.

According to a further aspect of the invention, the preparationaccording to the invention optionally contains a blood factor in theform of a zymogen or an active serine protease as a further component.Preferred further components are components having FEIB activity. Amongthem are, in particular, factor II, factor VII, factor IX, factor VIII,factor V and/or the active serine proteases thereof. Further componentscan also be phospholipids, Ca ions etc. According to a particularembodiment of the invention, the preparation according to the inventioncontains at least one further component having FEIB activity.

The preparation according to the invention can be provided as apharmaceutical preparation having factor Xa activity as a singlecomponent preparation or in combination with other factors as a multiplecomponent preparation.

Before processing to a pharmaceutical preparation, the purified proteinis subjected to the usual quality controls and brought into atherapeutically administrable form. In recombinant preparation, thepurified preparation is particularly tested for the absence of cellularand expression vector derived nucleic acids, preferably according to amethod as described in EP 0 714 987.

As, in principle, any biological material can be contaminated withinfectious germs, the preparation is optionally treated for inactivationor depletion of viruses in order to produce a safe preparation.

A further aspect of the invention refers to the use of a preparation asdescribed above in the preparation of a medicament. A medicamentcontaining a factor XΔ analogue according to the invention and acorrespondingly activated factor X analogue is particularly useful inthe treatment of patients suffering from blood coagulation disorderssuch as patients suffering from hemophilia or patients who havedeveloped inhibiting antibodies against the therapeutic agentadministered, e.g. against factor VIII or factor IX.

A further aspect of the invention relates to a method for thepreparation of the factor XΔ analogue and a preparation containing thefactor XΔ analogue according to the invention. The sequence encoding thefactor XΔ analogue is inserted into an appropriate expression system,and appropriate cells are transfected with the recombinant DNA.Preferably, permanent cell lines are established which express factor XΔanalogue. The cells are cultivated under optimal conditions for geneexpression, and factor X analogues are isolated either from a cellculture extract or from the cell culture supernatant. The recombinantmolecule can be further purified by all known chromatographic methods,such as anion or cation exchange, affinity or immunoaffinitychromatography or a combination thereof.

For the preparation of the factor XΔ analogues according to theinvention, the entire cDNA encoding the factor X is cloned in anexpression vector. This is effected according to generally known cloningtechniques. Subsequently, the nucleotide sequence encoding factor X ismodified such that the sequences encoding the amino acids Arg180 toArg234 are deleted and amino acids in the region between Gly173 andArg179, optionally Ile235, are modified such that a factor XΔ moleculeas described above can be produced. This is effected by geneticengineering techniques known from the state of the art, such as directedin vitro mutagenesis, deletion of sequences, e.g. by restrictiondigestion by endonucleases and insertion of other, changed sequences, orby PCR. The factor XΔ mutants thus prepared are then inserted into anexpression system appropriate for recombinant expression and areexpressed.

The factor XΔ analogues according to the invention can also be preparedby chemical synthesis.

The factor XΔ analogues are preferably produced by recombinantexpression. They can be prepared by means of genetic engineering withany usual expression systems, such as, for instance, permanent celllines or viral expression systems. Permanent cell lines are prepared bystable integration of the foreign DNA into the host cell chromosome of,e.g., vero, MRC5, CHO, BHK, 293, Sk-Hep1, particularly liver and kidneycells, or by an episomal vector derived, e.g., from the papilloma virus.Viral expression systems, such as, for instance, the vaccinia virus,baculovirus or retroviral systems, can also be employed. As cell lines,vero, MRC5, CHO, BHK, 293, Sk-Hep1, gland, liver and kidney cells aregenerally used. As eukaryotic expression systems, yeasts, endogenousglands (e.g. glands of transgenic animals) and other types of cells canbe used, too. Of course, transgenic animals can also be used for theexpression of the polypeptides according to the invention or derivativesthereof. For the expression of the recombinant proteins, CHO-DHFR⁻ cellshave proved particularly useful (Urlaub et al., Proc. Natl. Acad. Sci.,U.S.A., 77:4216-4220, 1980).

For the recombinant preparation of factor XΔ analogues according to thepresent invention, prokaryotic expression systems can be used, too.Systems allowing expression in E. coli or B. subtilis are particularlyuseful.

The factor XΔ analogues are expressed in the respective expressionsystems under control of a suitable promotor. For expression ineukaryotes, all known promoters are suitable, such as SV40, CMV, RSV,HSV, EBV, β-actin, hGH or inducible promoters, such as, for instance,hsp or metallothionein promotor. The factor X analogues are preferablyexpressed under control of the β-actin promotor in CHO-DHFR⁻ cells.

According to an embodiment of the invention, the method for preparingthe preparation of the invention comprises the steps of: providing a DNAencoding a factor XΔ analogue, transforming a cell with the recombinantDNA, expressing the factor X analogue, optionally in the presence of aprotease, isolating the factor X analogue, and optional purifying bymeans of a chromatographic method.

According to an embodiment of the process, the factor Xa analogue isdirectly isolated as a double chain molecule. A factor XΔ analoguehaving a modification allowing processing by a dibasic protease, such asfurin, is expressed in a cell, and the factor XΔ analogue is processedto double chain factor Xa analogue. The cell is preferably a cellexpressing a protease able to process, e.g. a dibasic protease, such asfurin or a derivative thereof. To improve or enhance processingefficiency, the cell can optionally be modified such that its proteaseexpression is enhanced. For instance, this can be effected byco-expression of a corresponding dibasic endoprotease, such asfurin/PACE, Kex2 or a derivative thereof. The factor XΔ analogueaccording to the invention can also be expressed in a cell having normalendogenous protease concentration, i.e. a suboptimal concentration forprocessing, resulting in incomplete processing into the double chainactive form. In this case, as long as single chain factor X analogue issecerned into the cell culture supernatant as described above,subsequent processing into factor Xa analogue is effected byco-cultivation with protease expressing cells or bringing into contactwith an optionally immobilized protease. The cell supernatant can alsobe pumped over a carrier matrix having protease bound thereto, thusyielding double chain factor Xa analogue in the eluate.

The factor Xa analogue thus obtained can subsequently be isolated,purified and optionally formulated as a pharmaceutical composition andstored stably until further use, as described above. The reactionconditions for the processing reaction and activation can be easilyoptimized by a person skilled in the art according to the experimentalsetup and the given basic conditions. For the contact time, the flowrate of the present reactants is of particular importance. It should bebetween 0.01 ml/min and 1 ml/min. Further important parameters aretemperature, pH value and eluation conditions. After passage, factor Xaanalogue can optionally be further purified by selective chromatography.It is particularly advantageous to conduct the process with proteasebound to a carrier, because when using a carrier, preferablychromatographic columns, the reaction setup allows an additionalpurification step.

According to an embodiment, activation is effected by a chromatographicstep, wherein protease is immobilized on a carrier. Purified singlechain factor XΔ analogue is conducted over a matrix having proteasebound thereto, and purified factor Xa analogue is isolated from theeluate.

According to an aspect of the invention, a preparation containing activefactor Xa analogue is obtained by subjecting factor XΔ analogue preparedas described above to a processing/activation step and furtherprocessing the activated polypeptide to a purified preparationoptionally formulated as a pharmaceutical composition.

According to a further aspect of the production of a preparationcontaining single chain factor XΔ analogue, e.g., the factor XΔ analoguehaving a processing sequence for a dibasic protease is expressed in acell having endoprotease deficiency. The cell is preferably deficient ina dibasic endoprotease, such as kexin, furin, PACE or homologousderivatives thereof. From such an endoprotease deficient mutant cell,factor XΔ analogue can be isolated as a single chain molecule. Factor XΔanalogues having a processing site for a serine protease can beexpressed in any conventional cell, including furin positive cells, andisolated as a single chain molecule.

A factor X analogue thus isolated and optionally purified issubsequently brought into contact with a protease selected from thegroup of endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2,PC4, PACE 4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa,factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or aderivative of these proteases, under conditions under which a singlechain factor X analogue is cleaved and activated to factor Xa analogue.

With the factor XΔ analogues according to the invention which areactivated by a process as described above to factor Xa analogues, apurified factor Xa analogue having high stability and structuralintegrity and being particularly free of inactive factor X/Xaintermediates is obtained.

The invention is described in more detail by the following Examples anddrawing figures, with the invention, however, not being restricted tothese particular examplary embodiments.

Example 1 describes the construction and expression of rfactor X;Example 2 describes the processing of rfactor X into heavy and lightchain by furin; Example 3 describes the processing of pro-factor X bymeans of immobilized protease; Example 4 describes the activity ofrfactor X processed in vitro; Example 5 describes the expression ofrfactor X in furin deficient cells; Example 6 describes the constructionand expression of rfactor XΔ analogues; Example 7 describes thedetermination of N-termini of the factor X processing products; Example8 describes the expression and characterization of the FX deletionmutant having the site Arg-Val-Thr-Arg/Ile (SEQ ID NO:101)(rFXΔ^(RVTR/I)); Example 9 describes in vitro activation of the proteinrFXΔ^(RVTR/I) by r-fufin derivatives.

The expression vectors were prepared by means of standard cloningtechniques (Maniatis et al., “Molecular Cloning”—A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., U.S.A., 1983).The preparation of DNA fragments by means of polymerase chain reaction(PCR) followed general methods (Clackson et al., 1991, PCR A practicalapproach. Ed. McPherson, Quirke, Taylor, p. 187-214).

EXAMPLE 1 Expression and Processing of Single Chain rFX to rFXLight/Heavy Chain

a. Preparation of the rFX Expression Vector

For the preparation of recombinant FX (rFX), the cDNA of FX was isolatedfrom a human liver lambda-cDNA-library as described by Messier et al.(1991, Gene 99:291-294). A DNA fragment was amplified from a positiveclone by means of PCR with oligonucleotide #2911(5′-ATTACTCGAGAAGCTTACCATGGGGCGCCCACTG-3′) (SEQ. ID No.1) as 5′-primerand oligonucleotide #2912 (5′-ATTACAATTGCTGCAGGGATCCAC-3′) (SEQ. ID. No.2) as 3′-primer, which DNA fragment contains the 1,467 kB FX codingsequence and 39 bp of the 3′-non-translated region, flanked by a XhoIcleavage site at the 5′-end and a MfeI cleavage site at the 3′-end. Inaddition, the sequence ACC was incorporated in front of the ATG of theFX by means of primer #2911 resulting in an optimal Kozak translationinitiation sequence. Subsequently, this PCR product was cloned asXhoI/MfeI fragment in the expression vector phact cleaved with SalI andEcoRI. The resulting expression plasmid was designated as phAct-rFX(FIG. 3). The expression vector phAct comprises the humanbeta-actin-promotor, 78 bp 5′UTR and the intron, a multiple cloningcleavage site, and the SV40 polyadenylation site.

b. Expression of rFX in CHO Cells

In order to establish a stable rFX expressing cell line, dhfr deficientCHO cells were co-transfected with the expression plasmid phAct-rFX andthe selection marker plasmid pSV-dhfr. For all further expression andfunction analyses, the cell cultures were incubated with serum freeselection medium in the presence of 10 μg/ml vitamin K for 24 hours. Theexpression of rFX in the resulting cell clones was detected by means ofthe amount of antigen (ELISA, Asserachrom, Boehringer Mannheim), andthen the recombinant protein was characterized with SDS-PAGE (FIGS. 4Aand B). As can be seen in the Western blot (FIG. 4A), in the initialclones and subclones thereof the recombinant FX protein is present inthe form of a light chain (LC) of 22 kD and a heavy chain (HC) ofapproximately 50 kD, which are identical with the plasmatic factor Xprotein. In addition, a protein band is visible at 75 kD, whichcorresponds to the single chain (SC) molecule and the presence of whichin FX transfected CHO cells (Wolf et al., J. Biol. Chem.266:13726-13730, 1991) and in human plasma (Fair et al., Blood64:194-204, 1984) has been described. For the preparation of highlyexpressing clones, the initial clones were amplified with increasingamounts of methotrexate and subsequently subcloned to stabilization.Expression could be increased from about 200-500 ng/10 E6 cells or 1μg/ml, respectively, to 78 μg/10 E6 cells or 120 μg/ml, respectively,per 24 hours. Western blot analysis of these highly expressing cellclone supernatants (FIGS. 4B and 5A, lane 2) shows enrichment of thesingle chain rFX molecule and the presence of additional forms of thelight chain. Besides the 22 kD form of the light chain, whichcorresponds to the plasmatic form (completely carboxylated and withoutpropeptide) there are three further light chain variants of about 21 kD,22.5 kD, and 20 kD present. By means of N-terminal sequencing of therecombinant material, the heterogeneity of the light chain in theseclones was determined as a result of incomplete cleavage of thepropeptide (here: about 50% of the rFX material) and hypocarboxylation(here: about 50% of the rFX). The 21 kD protein is a hypocarboxylated,propeptide containing form, and the 20 kD protein is a hypocarboxylated,propeptide-free form of the light chain, while the 22.5 kD bandrepresents the fully carboxylated, but propeptide containing LC form.

EXAMPLE 2 Processing of Single Chain rFX in rFX Light/Heavy Chain byr-Furin Derivatives

Due to the similarity of the cleavage sites of factor Xpropeptide/N-terminus of the light chain (RVTR↓A; SEQ ID NO:129) and oflight/heavy chain (RRKR↓S; SEQ ID NO:130) to the furin consensusdetection sequence (RXK/RR↓X; SEQ ID NO:131), it was possible to improvein vitro processing of single chain as well as propeptide containing rFXmolecules by r-furin derivatives. In the literature, proteases aresuspected for the two processing steps, which, however, are not furin(Rehemtulla et al., 1992, Blood 79:2349-2355; Wallin et al., 1994,Thromb. Res. 1994:395-403).

Cell culture supernatants of CHO-rFX and CHO-rfurin ΔTM6xHis (patentapplication EP 0 775 750) as well as CHO-rFX and non-transfected CHO (asnegative control) were mixed at a ratio of 1:1 and incubated at 37° C.Aliquots of the reaction mixtures were tested for processed rFX beforeincubation (t=0) and after various incubation periods (t=2, 4, 6, hours)by means of Western blot analysis (FIGS. 5A and 5B). The rFX wasdetected in the cell culture supernatants by means of an anti-human FXantiserum (FIG. 5A) and a monoclonal antibody specific for the lightchain of FX (FIG. 5B).

Contrary to the CHO-rFX/CHO mixture, CHO-rFX/CHO-rfurin shows almostcomplete processing already after 2 hours of incubation at 37° C. (FIG.5A, lane 7; FIG. 5B, lane 8). Single chain rFX is largely reacted to thelight and heavy chain forms. In the area of the light chain, only theprocessed propeptide-free forms of 22 kD (carboxylated form) and 20 kD(hypocarboxylated form) were found at a ratio of about 50:50. Byoptimizing cell culture conditions, this ratio can be improved in favorof the carboxylated form. Correct cleavage of the pro-sequence betweenArg-1 and Ala+1 and homogeneity of the N-terminus of the light chainwere determined by means of N-terminal sequencing. In the controlexperiment, wherein CHO-rFX was mixed with CHO-supernatants, no changein the rFX band pattern is visible even after 6 hours of incubation(FIG. 5A, lane 5; FIG. 5B, lane 6). This proves that r-furin in thesupernatant of CHO cells is biologically active and can process thepropeptide as well as the heavy/light chain of rFX.

EXAMPLE 3 Processing of Factor X by Means of Chelate-tentacle GelImmobilized r-Furin

To determine whether a substrate can be cleaved by a column-boundr-furin derivative, a study was conducted as to whether in anexperimental setup Fractogel EMD® tentacle gel (Merck) can be usedinstead of Ni²⁺-NTA agarose as column matrix. As the metal ions arefarther apart from the actual column matrix than the Ni²⁺-NTA agarose,an improved sterical access to the bound r-furin derivative might beachieved. In the present setup, pro-factor X was processed by tentaclegel bound r-furin derivative:

Fractogel EMD® tentacle gel was loaded with Ni²⁺ ions according to theproducer's instructions and equilibrated with fresh serum-free cellculture medium. Subsequently, the column was loaded with serum-freeCHO-r-furin derivative supernatant. Washing steps were carried out withserum-free cell culture medium containing increasing imidazoleconcentrations up to 40 mM. Then pro-factor X was passed over the columnas serum-free CHO supernatant. Processing of pro-factor X to doublechain factor X was detected in the effluent of the column by means ofWestern blot analysis with specific factor X antiserum.

EXAMPLE 4 Activity of Recombinant Factor X Processed In Vitro

Recombinant factor X precursor was incubated with and without r-furin at4° C. At different times, samples were taken and frozen at −20° C. Afterthe incubation was completed (after 4 days), all samples were tested forFX activity using a FX Coatest Kit (Chromogenix). 50 μl of eachsupernatant were mixed with 50 μl FX deficient human plasma, and rFX wasreacted with snake venom (RVV) to rFXa in the presence of CaCl₂according to the producer's instructions, rFXa then hydrolyzes thechromogenic substrate (S-2337) and leads to the release ofyellow-coloured paranitroaniline. As the amount of rFXa and theintensity of the colour are proportionate to each other, the amount ofrFX/ml cell culture supernatant which can be activated to rFXa can bedetermined by means of a calibration line interpolated from values of aplasma dilution series. Using these results and the known amount of rFXantigen (ELISA data), the proportion of rfactor X activated to factor Xacan be calculated in %. The results are presented in table 1.

In order to exclude nonspecific, proteolytic activity in CHO andCHO-r-furin supernatants, the mixture of these two cell culturesupernatants was tested, too.

Even after 4 days, CHO-rFX incubated with CHO supernatants (withoutr-furin) as control displayed no substantial change in rFXa activity,which was about 800 mU/ml and corresponded to 50% to 60% of functionalrFX due to experimental variations. When, in comparison, CHO-rFX wasincubated with CHO-r-furin, rFX activity increased steadily duringincubation, rising from about 60% (T=0) to 86% (table 1). This provesthat in vitro processing of CHO-rFX from highly expressing clones usingr-furin derivative substantially improves the proportion of rFX that canbe activated to functional rFXa.

TABLE 1 amount of functional incubation activity antigen portion of(days) (mU) (μg/ml) rFX (%) CHO-rFX + 0 814 14 58 CHO 1 847 14 61 2 83514 60 3 790 14 56 4 763 14 55 CHO-rFX + 0 853 14 61 CHO-rFurin 1 1018 1473 2 1099 14 79 3 1135 14 81 4 1198 14 86 CHO + 0 CHO-rFurin Plasma FX585 500 mU

EXAMPLE 5 Expression of Recombinant Factor X in Furin Deficient Cells

As shown in the previous Examples, in the case of factor X precursorprotein, furin mediates propeptide cleavage as well as cleavage of thesingle chain to light/heavy chain in vitro. This suggests that thesesteps are also effected endogenously in the cell by ubiquitous furinwith varying efficiency depending on the amount of expressed rfactor X.This in turn leads to the production of a mixture of heterogenousrfactor X forms.

One way to prepare a form of rfactor X molecules which is as homogeneousas possible and also stable is to prevent cleavage of rfactor X byendogenous proteases, particularly furin, and thus to producefunctionally inactive rfactor X precursors (which can be transformedinto its functionally active form later by means of downstreamprocessing, ideally directly before use). This process will beparticularly useful in the preparation of FX deletion mutants containinga furin cleavage site instead of the original activation site. In theseconstructs, such a recombinant rFX mutant in vivo can be activated byendogenous furin and lead to the secretion of activated, more instablerFX forms. Degradation of these forms by CHO proteases, e.g. under cellculture conditions of high cell lysis, during storage of the cellculture supernatants or the purifying process could result in inactivedegradation products (Wolf et al., 1991).

This aim can, for instance, be achieved by supplementing the cellculture medium with agents which can reduce or prevent intracellularfurin activity.

Another way is to use cells which are furin deficient a priori (Möhringet al., 1983, Infect. Immun. 41:998-1009; Ohnishi et al., 1994, J.Virol. 68:4075-4079; Gordon et al., 1995, Infect. Immun. 63:82-87).

For this purpose, a furin deficient CHO cell clone FD11(Gordon et al.,1995, Infect. Immun. 63:82-87) was co-transfected with 20 μg phAct-FXand 1 μg pUCSV-neo (containing the neomycin resistance gene in the pUCvector under control of the SV40 promotor). In order to obtain stableclones, the medium was supplemented with 0,8 μg G418/ml. Comparingsecerned rfactor X molecules in serum free supernatants of a furincontaining and a furin deficient CHO clone, Western blot shows thatrfactor X precursor is not processed in the furin deficient cells andonly single chain factor X precursor is present (FIG. 6); in contrast,rfactor X is still completely processed by “normal” cells with modestexpression, but is processed only to a very limited extent with higherexpression in spite of endogenous furin. Due to the low degree of rFXexpression of the cell clone used, the light chain of rfactor X here isnot visible in the blot.

EXAMPLE 6 Preparation of Factor XΔ Analogues (at Present, the ApplicantRegards this as the Best Mode for Carrying Out the Invention)

6.1. Construction of Expression Plasmids for the Preparation of FXDeletion Mutants

Factor X deletion mutants differ from the factor X wild type sequence inthe deletion of the app. 4.5 kDa activation peptides between amino acid180 and 234. In addition, various cleavage sites were introduced intothe C-terminus of the light chain and/or the N-terminus of the heavychain by means of mutagenesis, which sites function to activate thesingle chain factor X molecule resulting therefrom to the activatedpolypeptide. Expression plasmids for these factor X deletion mutants areall derived from phAct-FX (described in Example 1).

In order to simplify the cloning of factor X deletion mutants, theHindIII-NaeI DNA fragment from plasmid phAct-FX, which comprises thefactor X encoding region from position +1 to +1116, was inserted intothe HindIII/SmaI restriction cleavage sites of plasmid pUC19. Theresulting plasmid was designated as pUC/FX. In order to delete theactivation peptide and to incorporate new cleavage sites, e.g. furin,FXIa, FXIIa, FXa, FIIa cleavage sites, the Bsp120I/BstXI FX DNA fragmentfrom the pUC/FX vector was replaced by synthetic oligonucleotides. Inorder to incorporate a thrombin or FXIa cleavage site, theBstXI-3′-overlap was smoothened by mung bean nuclease, so that aminoacid Ile at position 235 could be exchanged, too. Subsequently, thedeleted factor X DNA fragments were cloned in plasmid pACT-FX viaHindIII-AgeI.

In order to prepare the Asp-Phe-Thr-Arg/Val (SEQ ID NO:132) FXIacleavage site, the oligonucleotide sense #0009 (5′-GG CCC TAC CCC TGTGGG AAA CAG GAC TTC ACC AGG GTG-3′) (SEQ ID NO:3) and theoligonucleotide antisense #0010 (5′-CAC CCT GGT GAA GTC CTG TTT CCC ACAGGG GTA G-3′) (SEQ ID NO:4) were used and inserted into the Bsp120I andthe mung bean nuclease treated BstXI sites. Thus, the amino acids fromposition 176 to 178 and 235 were mutated into Asp-Phe-Thr and Val (FIG.2A, panel A).

In order to prepare the Arg/Thr FIIa cleavage site, the oligonucleotidesense #0011 (5′-GG CCC TAC CCC TGT GGG AAA CAG ACC CTG GAA CGG ACC-3′)(SEQ ID NO:5) and the oligonucleotide antisense #0012 (5′-GGT CCG TTCCAG GGT CTG TTT CCC ACA GGG GTA G-3′) (SEQ ID NO:6) were used andinserted into the Bsp120I and the mung bean nuclease treated BstXIsites. Thus, the amino acid lie at position 235 was mutated into Thr(FIG. 2A, panel B).

In order to prepare the Ile-Lys-Pro-Arg/Ile (SEQ ID NO:133) FXIIacleavage site, the oligonucleotide sense #0013 (5′-GG CCC TAC CCC TGTGGG AAA CAG ATC AAG CCC AGG ATC-3′) (SEQ ID NO:7) and theoligonucleotide antisense #0014 (5′-CT GGG CTT GAT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:8) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of position 176 to 178 were mutatedinto Ile-Lys-Pro (FIG. 2A, panel C).

In order to prepare the Ser-Met-Thr-Arg/Ile (SEQ ID NO:134) kallikreincleavage site, the oligonucleotide sense #0015 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGC ATG ACC AGG ATC-3′) (SEQ ID NO:9) and theoligonucleotide #0016 (5′-CT GGT CAT GCT CTG TTT CCC ACA GGG GTA G-3′)(SEQ ID NO:10) were used and inserted into the Bsp120I and BstXI sites.Thus, the amino acids of position 176 to 178 were mutated intoSer-Met-Thr (FIG. 2A, panel D).

In order to prepare the Met-Lys-Thr-Arg/Ile (SEQ ID NO:135) FXa cleavagesite, the oligonucleotide sense #0033 (5′-GG CCC TAC CCC TGT GGG AAA CAGATG AAA ACG AGG ATC-3′) (SEQ ID NO:11) and the oligonucleotide antisense#0034 (5′-CT CGT TTT CAT CTG TTT CCC ACA GGG GTA G-3′) (SEQ ID NO:12)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of position 176 to 178 were mutated from Thr-Leu-Glu intoMet-Lys-Thr (FIG. 2A, panel E).

In order to prepare the Ile-Glu-Gly-Arg/Ile (SEQ ID NO:136) FXa cleavagesite, the oligonucleotide sense #0035 (5′-GG CCC TAC CCC TGT GGG AAA CAGATC GAG GGA AGG ATC-3′) (SEQ ID NO:13) and the oligonucleotide antisense#0036 (5′-CT TCC CTC GAT CTG TTT CCC ACA GGG GTA G-3′) (SEQ ID NO:14)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids in position 176 to 178 were mutated from Thr-Leu-Glu intoIle-Glu-Gly (FIG. 2A, panel F).

In order to prepare the Arg-Arg-Lys-Arg/Ile (SEQ ID NO:137) furincleavage site, the oligonucleotide sense #0017 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGG AGG AAG AGG ATC-3′) (SEQ ID NO:15) and theoligonucleotide antisense #0018 (5′-CT CTT CCT CCT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:16) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids in positions 176 to 178 were mutatedinto Arg-Arg-Lys (FIG. 2A, panel G).

In order to prepare the Arg-Val-Arg-Arg/Ile (SEQ ID NO:138) furincleavage site, the oligonucleotide sense #0019 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGG GTG AAG AGG ATC-3′) (SEQ ID NO:17) and theoligonucleotide antisense #0020 (5′-CT CCT CAC CCT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:18) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of positions 176 to 178 were mutatedinto Arg-Val-Arg (FIG. 2A, panel G).

In order to prepare the Arg-Arg-Arg-Arg/Ile (SEQ ID NO:139) furincleavage site, the oligonucleotide sense #0021 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGG AGG AGG AGG ATC-3′) (SEQ ID NO:19) and theoligonucleotide antisense #0022 (5′-CT CCT CCT CCT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:20) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of positions 176 to 178 were mutatedinto Arg-Arg-Arg (FIG. 2A, panel G).

In order to prepare the Arg-Pro-Lys-Arg/Ile (SEQ ID NO:140) furincleavage site, the oligonucleotide sense #0023 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGG CCC AAG AGG ATC-3′) (SEQ ID NO:21) and theoligonucleotide antisense #0024 (5′-CT CTT GGG CCT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:22) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of positions 176 to 178 were mutatedinto Arg-Pro-Lys (FIG. 2A, panel G).

In order to prepare the Ile-Arg-Lys-Arg/Ile (SEQ ID NO:141) furincleavage site, the oligonucleotide sense #0025 (5′-GG CCC TAC CCC TGTGGG AAA CAG ATC AGG A AG AGG ATC-3′) (SEQ ID NO:23) and theoligonucleotide antisense #0026 (5′-CT CTT CCT GAT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:24) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of positions 176 to 178 were mutatedinto Ile-Arg-Lys (FIG. 2A, panel G).

In order to prepare the Arg-Ser-Lys-Arg/Ile (SEQ ID NO:142) furincleavage site, the oligonucleotide sense #0027 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGG AGC AAG AGG ATC-3′) (SEQ ID NO:25) and theoligonucleotide antisense #0028 (5′-CT CTT GCT CCT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:26) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of positions 176 to 178 were mutatedinto Arg-Ser-Lys (FIG. 2A, panel G).

In order to prepare the Arg-Val-Thr-Arg/Ile (SEQ ID NO:101) furincleavage site, the oligonucleotide sense #0029 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGG GTC ACG AGG ATC-3′) (SEQ ID NO:27) and theoligonucleotide antisense #0030 (5′-CT CGT GAC CCT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:28) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of positions 176 to 178 were mutatedinto Arg-Val-Thr (FIG. 2A, panel G).

In order to prepare the Arg-Leu-Lys-Arg/Ile (SEQ ID NO:143) furincleavage site, the oligonucleotide sense #0031 (5′-GG CCC TAC CCC TGTGGG AAA CAG AGG CTG AAA AGG ATC-3′) (SEQ ID NO:29) and theoligonucleotide antisense #0032 (5′-CT TTT CAG CCT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:30) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids of positions 176 and 178 were mutatedinto Arg and Lys (FIG. 2A, panel G).

In order to prepare the Pro-Gln-Gly-Arg/Ile (SEQ ID NO:144) FXa cleavagesite, the oligonucleotide sense #0037 (5′-GG CCC TAC CCC TGT GGG AAA CAGCCC CAA GGA AGG ATC-3′) (SEQ ID NO:31) and the oligonucleotide antisense#0038 (5′-CT TCC TTG GGG CTG TTT CCC ACA GGG GTA G-3′) (SEQ ID NO:32)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids in positions 176 to 178 were mutated from Thr-Leu-Glu intoPro-Gln-Gly (FIG. 2A, panel H).

In order to prepare the Thr-Ser-Thr-Arg/Ile (SEQ ID NO:145) FXIIacleavage site, the oligonucleotide sense #0039 (5′-GG CCC TAC CCC TGTGGG AAA CAG ACG AGC ACG AGG ATC-3′) (SEQ ID NO:33) and theoligonucleotide antisense #0040 (5′-CT CGT GCT CGT CTG TTT CCC ACA GGGGTA G-3′) (SEQ ID NO:34) were used and inserted into the Bsp120I andBstXI sites. Thus, the amino acids in positions 177 and 178 were mutatedinto Ser-Thr (FIG. 2A, panel 1).

In order to prepare an Arg/Ile trypsin cleavage site, theoligonucleotide #0041 (5′-GG CCC TAC CCC TGT GGG AAA CAG ACC CTG GAA CGGATC-3′) (SEQ ID NO:35) and the oligonucleotide antisense #0042 (5′-CGTTC CAG GGT CTG TTT CCC ACA GGG GTA G-3′) (SEQ ID NO:36) were used andinserted into the Bsp120I and BstXI sites (FIG. 2A, panel J).

The resulting expression plasmids (see FIG. 3) comprise the humanbeta-actin-promotor, 78 bp of 5′UTR, the beta-actin-intron, the modifiedfactor X sequence, and 39 bp of the 3′UTR and the SV40 polyadenylationsite.

6.2. Construction of Expression Plasmids for the Preparation of FXβAnalogue

These constructs were derived from the factor XΔ analogue constructsdescribed above by introducing a TGA stop codon into position 470. Theamino acids from position 457 to the stop codon were removed by SpeI andpartial BstEII digestion and replaced by the oligonucleotide pair #0003(5′ GTC ACC GCC TTC CTC AAG TGG ATC GAC AGG TCC ATG AAA ACC AGG TGAA-3′) (SEQ ID NO:37) and #0004 (5′-CTA GTT CAC CTG GTT TTC ATG GAC CTGTCG ATC CAC TTG AGG AAG GCG-3′) (SEQ ID NO:38). FIG. 7 is a schematicrepresentation of the factor XΔβ analogue constructs. In order to simplythe figure, all factor XΔβ analogues are represented as a generalconstruct wherein the variable amino acids in the cleavage site regionare designated as a shaded “X”.

6.3. Construction of Expression Plasmids for the Production of FXΔαAnalogue

By activating factor X by cleaving off the 4.5 kDa activation peptide atthe N-terminal end of the heavy chain, the factor Xaα form is generated.This form is subsequently reacted to the FXaβ form by autoproteolyticactivity and cleavage of the C-terminus of the heavy chain betweenArg469 and Gly470. For the preparation of factor X expression plasmidsleading to the production of factor XΔ analogues, which will be presentafter activation exclusively in the Fxaα form having intact β-peptide,the amino acid Arg469 was mutated to Lys so that the C-terminal regionof the heavy chain can not be processed any more.

For this purpose, the DNA sequence of factor X encoding the C-terminalamino acid sequence was removed from position 1363 to the stop signal bypartial BstEII-SpeI digestion and replaced by two ligatedoligonucleotide pairs. Oligonucleotide #0005 (5′-GTC ACC GCC TTC CTC AAGTGG ATC GAC AGG TCC ATG AAA ACC AAG GGC TTG CCC AAG-3′) (SEQ ID NO:39)and oligonucleotide #0006 (5′-TTG GCC TTG GGC AAG CCC TTG GTT TTC ATGGAC CTG TCG ATC CAC TTG AGG AAG GCG-3′) (SEQ ID NO:40) were ligated witholigonucleotide #0007 (5′-GCC AAG AGC CAT GCC CCG GAG GTC ATA ACG TCCTCT CCA TTA AAG TGA GAT CCC A-3′) (SEQ ID NO:41) and oligonucleotide#0008 (5′-CTA GTG GGA TCT CAC TTT AAT GGA GAG GAC GTT ATG ACC TCC GGGGCA TGG CTC-3′) (SEQ ID NO:42). The mutation of amino acid Arg469 isintroduced by the oligonucleotide pair #0005-0006. FIG. 7 is a schematicrepresentation of the FXΔ analogues.

EXAMPLE 7 Determination of the N-termini of Factor X and ProcessingProducts With and Without r-Furin

Recombinant factor X was expressed in CHO cells having endogenous furin,as described in Example 1, and in furin deficient cells, as described inExample 5. rFactor X was isolated from cell culture supernatant ofhighly expressing CHO-rFX clones, which was a) not pre-treated, b)incubated at 37° C. for 12 hours and c) pre-treated with CHO-r-furinsupernatant at 37° C. for 12, hours, as well as from cell culturesupernatant of CHO-FD11-rFX clones which was d) not pre-treated and e)pre-treated with CHO-r-furin supernatant at 37° C. for 12 hours. Theterminal N-terminal amino acids of factor X and the processing productsof the individual reaction mixtures a) to e) were determined by Edmananalysis. FIG. 8 is a schematic representation of the results.

rFactor X from highly expressing CHO cells is present in the form of themature heavy and light chains as well as in the single chain form,partly still containing propeptide. After incubation of these cellculture supernatants for 12 hours at 37° C. (b), additional faultyN-termini of the rFX light chain having 3 additional amino acidsVal38-Thr39-Arg40 are formed, as described by Wolf et al. (1991, J. Bio.Chem. 266:13726-13730). These cryptic ends are also found whensequencing rFX material from non-pre-treated CHO-FD11 cells (d). Thisobservation shows that the formation of these faulty N-termini can beprevented by reasonable conditions, i.e. cell culture conditions,storage and purifying processes in order to minimize rFX proteolysis byCHO proteases.

Contrary to the purified material from CHO cells (a and b), rFX fromnon-amplified, furin deficient cells (d) is only present in the form ofunprocessed single chain precursors. N-terminal sequences correspondingto the propeptide portion are not found, either. This shows that singlechain rFX precursor is not processed any more to light/heavy chain infurin deficient CHO cells (d), which suggests a central role of theendoprotease furin in this processing step in vivo. In addition, itshows that rFX molecules containing propeptide are also processed infurin deficient CHO cells, i.e. that furin does not play an essentialrole in this processing step in vivo. After incubation of rFX from CHOcells (c) and CHO-FD11 cells (e) in the presence of furin, only lightand heavy chains having correct N-termini are found. This proves thatthe single chain FX precursors as well as the propeptide containing rFXmolecules are reacted to homogenous, mature factor X by in vitroprocessing. Thus, factor X processed in the presence of furin exhibitsexceptional structural integrity.

EXAMPLE 8 Expression and Characterization of the Recombinant FX DeletionMutant Having the Furin Cleavage Site Arg-Val-Thr-Arg/Ile (SEQ IDNO:101) (FXΔ^(RVTR/I))

The expression plasmid encoding the FX deletion mutant having thecleavage site Arg-Val-Thr-Arg/Ile (SEQ ID NO:101) (FXΔ^(RVTR/I)) wasco-transfected with the selection marker pSV/dhfr in dhfr deficient CHOcells as described in Example 1. The recombinant protein FXΔ^(RVTR/I)from permanent CHO clones was characterized by means of Western blotanalysis. As can be seen in FIG. 9, lane 4, the recombinant protein ispresent in the form of a double band of approximately 56 and 50 kD. NoFX reactive material is detectable in the cell culture supernant ofnon-transfected CHO cells (lane 2). According to these results, it isimpossible that these protein bands result from impurities of theanalyzed supernatants of wild type FX from the residues of bovine serumin the cell culture medium. Therefore, the double band is possiblycaused by different post-translational modifications, e.g. the presenceof the propeptide or different glycosylation of the rFXΔ^(RVTR/I)molecule.

The cleavage site Arg-Val-Thr-Arg/Ile (SEQ ID NO:101) inserted into thisconstruct is identical with the propeptide cleavage site of the wildtype FX molecule, which is efficiently recognized and cleaved in vivo bya CHO endoprotease (see Example 7). The Western blot analysis shows noadditional 35 kD and 31 kD heavy FX molecules, which would correspond tothe activated α- and β-forms of the rFXΔ^(RVTR/I) heavy chain. Theseresults show that either the amount of endoprotease is not sufficient toactivate the protein or/and that the cleavage site Arg-Val-Thr-Arg/Ile(SEQ ID NO:101) is not or not effectively recognized and cleaved in vivoin the present sequence environment. Consequently, rFXΔ^(RVT/I) ispractically only present in the single chain form.

EXAMPLE 9 Activation of the Recombinant rFXΔ^(RVTR/I) Protein by Meansof Recombinant Furin Derivatives In Vitro

Although the cleavage site Arg-Val-Thr-Arg/Ile (SEQ ID NO:101) in therFX propeptide is recognized in vivo by a protease other than furin,Example 2 proves that this sequence is cleaved very efficiently andcorrectly by an r-furin derivative in vitro.

Mixing experiments were carried out in order to test the ability ofrFXΔ^(RVTR/I) protein to be activated by r-furin in vitro. Cell culturesupernatant from CHO-FXΔ^(RVTR/I) cells were mixed with purified r-furinderivative r-furinΔCys-spacer-10xHis (see patent application EP-0 775750-A2) in the presence of 20 mM Hepes pH 7.0, 150 mM NaCl, 4 mM CaCl₂and 0.1% BSA at a ratio of 1:1. In control experiment, theCHO-rFXΔ^(RVTR/I) supernatant was mixed only with BSA containing bufferat the same ratio. The addition of BSA is meant to stabilize theenzymatic activity of the r-furin derivative and the activatedrFXΔ^(RVTR/I) products consequently formed. Aliquots of the reactionmixture were tested before and after an incubation period of 6, 24, 48and 72 hours (t=0, t=6, t=24, t=48, t=72) at 37° C. for rFXΔ^(RVTR/I)processing by means of Western blot analysis (FIGS. 10A and 10B). In themixing experiment without r-furin addition (FIG. 10B), no change in theband pattern is visible during the incubation period (lanes 4 to 9). Dueto the presence of BSA in the reaction mixtures, only the lighterrFXΔ^(RVTR/I) molecules (50 kD) are easily visible, because the 56 kDheavy molecules are covered by the BSA band. In the presence of ther-furin derivative (FIG. 10A), a 35 kD protein band appears alreadyafter 6 hours of incubation (lane 5), which corresponds to the α-form ofthe FX heavy chain (cf. lane 9). This protein accumulates in the courseof incubation and is subsequently reacted to the proteolytic β-form, asalready known in the case of plasma FX, which β-form forms byproteolytic conversion from the α-form (lanes 7 and 8). Light chains of22 kD and 20 kD appear parallel to the detection of the activated formsof the heavy chains, which light chains were identified as propeptidefree, carboxylated LC2 form (corresponding to the actually functionalform) or as propeptide free, hypocarboxylated LC4 form of the lightchain in Example l.b. The presence of the hypocarboxylated LC4 formproves that the post-translational modification mechanisms are limitedin the analyzed CHO clones. Although the 50 kD bond appears to beunchanged, while apparently the 56 kD form is directly degraded tolight/heavy chains, in fact the 56 kD molecule at first is convertedinto the 50 kD form, and only subsequently is cleaved into a light and aheavy chain. This is due to the presence of the propeptide in the 56 kDmolecule which at first is removed by forming the 50 kD form.

This proves that the rFXΔ^(RVTR/I) construct can be activated in vitroby r-furin derivatives via an inserted Arg-Val-Thr-Arg/Ile (SEQ IDNO:101) cleavage site and the resulting processing products of therFXΔ^(RVTR/I) construct correspond to those of plasma FXa in size. Theemergence of FXΔβ, which is formed due to autoproteolytic processing ofFXΔα, shows the functionality of the rFXΔ^(RVTR/I) molecule.

145 34 base pairs nucleic acid single linear DNA 1 ATTACTCGAG AAGCTTACCATGGGGCGCCC ACTG 34 24 base pairs nucleic acid single linear DNA 2ATTACAATTG CTGCAGGGAT CCAC 24 38 base pairs nucleic acid single linearDNA 3 GGCCCTACCC CTGTGGGAAA CAGGACTTCA CCAGGGTG 38 34 base pairs nucleicacid single linear DNA 4 CACCCTGGTG AAGTCCTGTT TCCCACAGGG GTAG 34 38base pairs nucleic acid single linear DNA 5 GGCCCTACCC CTGTGGGAAACAGACCCTGG AACGGACC 38 34 base pairs nucleic acid single linear DNA 6GGTCCGTTCC AGGGTCTGTT TCCCACAGGG GTAG 34 38 base pairs nucleic acidsingle linear DNA 7 GGCCCTACCC CTGTGGGAAA CAGATCAAGC CCAGGATC 38 30 basepairs nucleic acid single linear DNA 8 CTGGGCTTGA TCTGTTTCCC ACAGGGGTAG30 38 base pairs nucleic acid single linear DNA 9 GGCCCTACCC CTGTGGGAAACAGAGCATGA CCAGGATC 38 30 base pairs nucleic acid single linear DNA 10CTGGTCATGC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 11 GGCCCTACCC CTGTGGGAAA CAGATGAAAA CGAGGATC 38 30 base pairsnucleic acid single linear DNA 12 CTCGTTTTCA TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 13 GGCCCTACCC CTGTGGGAAACAGATCGAGG GAAGGATC 38 30 base pairs nucleic acid single linear DNA 14CTTCCCTCGA TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 15 GGCCCTACCC CTGTGGGAAA CAGAGGAGGA AGAGGATC 38 30 base pairsnucleic acid single linear DNA 16 CTCTTCCTCC TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 17 GGCCCTACCC CTGTGGGAAACAGAGGGTGA GGAGGATC 38 30 base pairs nucleic acid single linear DNA 18CTCCTCACCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 19 GGCCCTACCC CTGTGGGAAA CAGAGGAGGA GGAGGATC 38 30 base pairsnucleic acid single linear DNA 20 CTCCTCCTCC TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 21 GGCCCTACCC CTGTGGGAAACAGAGGCCCA AGAGGATC 38 30 base pairs nucleic acid single linear DNA 22CTCTTGGGCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 23 GGCCCTACCC CTGTGGGAAA CAGATCAGGA AGAGGATC 38 30 base pairsnucleic acid single linear DNA 24 CTCTTCCTGA TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 25 GGCCCTACCC CTGTGGGAAACAGAGGAGCA AGAGGATC 38 30 base pairs nucleic acid single linear DNA 26CTCTTGCTCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 27 GGCCCTACCC CTGTGGGAAA CAGAGGGTCA CGAGGATC 38 30 base pairsnucleic acid single linear DNA 28 CTCGTGACCC TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 29 GGCCCTACCC CTGTGGGAAACAGAGGCTGA AAAGGATC 38 30 base pairs nucleic acid single linear DNA 30CTTTTCAGCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 31 GGCCCTACCC CTGTGGGAAA CAGCCCCAAG GAAGGATC 38 30 base pairsnucleic acid single linear DNA 32 CTTCCTTGGG GCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 33 GGCCCTACCC CTGTGGGAAACAGACGAGCA CGAGGATC 38 30 base pairs nucleic acid single linear DNA 34CTCGTGCTCG TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 35 GGCCCTACCC CTGTGGGAAA CAGACCCTGG AACGGATC 38 30 base pairsnucleic acid single linear DNA 36 CGTTCCAGGG TCTGTTTCCC ACAGGGGTAG 30 49base pairs nucleic acid single linear DNA 37 GTCACCGCCT TCCTCAAGTGGATCGACAGG TCCATGAAAA CCAGGTGAA 49 48 base pairs nucleic acid singlelinear DNA 38 CTAGTTCACC TGGTTTTCAT GGACCTGTCG ATCCACTTGA GGAAGGCG 48 57base pairs nucleic acid single linear DNA 39 GTCACCGCCT TCCTCAAGTGGATCGACAGG TCCATGAAAA CCAAGGGCTT GCCCAAG 57 57 base pairs nucleic acidsingle linear DNA 40 TTGGCCTTGG GCAAGCCCTT GGTTTTCATG GACCTGTCGATCCACTTGAG GAAGGCG 57 55 base pairs nucleic acid single linear DNA 41GCCAAGAGCC ATGCCCCGGA GGTCATAACG TCCTCTCCAT TAAAGTGAGA TCCCA 55 54 basepairs nucleic acid single linear DNA 42 CTAGTGGGAT CTCACTTTAA TGGAGAGGACGTTATGACCT CCGGGGCATG GCTC 54 1467 base pairs nucleic acid single linearcDNA CDS 1...1467 Factor X 43 ATG GGG CGC CCA CTG CAC CTC GTC CTG CTCAGT GCC TCC CTG GCT GGC 48 Met Gly Arg Pro Leu His Leu Val Leu Leu SerAla Ser Leu Ala Gly 1 5 10 15 CTC CTG CTG CTC GGG GAA AGT CTG TTC ATCCGC AGG GAG CAG GCC AAC 96 Leu Leu Leu Leu Gly Glu Ser Leu Phe Ile ArgArg Glu Gln Ala Asn 20 25 30 AAC ATC CTG GCG AGG GTC ACG AGG GCC AAT TCCTTT CTT GAA GAG ATG 144 Asn Ile Leu Ala Arg Val Thr Arg Ala Asn Ser PheLeu Glu Glu Met 35 40 45 AAG AAA GGA CAC CTC GAA AGA GAG TGC ATG GAA GAGACC TGC TCA TAC 192 Lys Lys Gly His Leu Glu Arg Glu Cys Met Glu Glu ThrCys Ser Tyr 50 55 60 GAA GAG GCC CGC GAG GTC TTT GAG GAC AGC GAC AAG ACGAAT GAA TTC 240 Glu Glu Ala Arg Glu Val Phe Glu Asp Ser Asp Lys Thr AsnGlu Phe 65 70 75 80 TGG AAT AAA TAC AAA GAT GGC GAC CAG TGT GAG ACC AGTCCT TGC CAG 288 Trp Asn Lys Tyr Lys Asp Gly Asp Gln Cys Glu Thr Ser ProCys Gln 85 90 95 AAC CAG GGC AAA TGT AAA GAC GGC CTC GGG GAA TAC ACC TGCACC TGT 336 Asn Gln Gly Lys Cys Lys Asp Gly Leu Gly Glu Tyr Thr Cys ThrCys 100 105 110 TTA GAA GGA TTC GAA GGC AAA AAC TGT GAA TTA TTC ACA CGGAAG CTC 384 Leu Glu Gly Phe Glu Gly Lys Asn Cys Glu Leu Phe Thr Arg LysLeu 115 120 125 TGC AGC CTG GAC AAC GGG GAC TGT GAC CAG TTC TGC CAC GAGGAA CAG 432 Cys Ser Leu Asp Asn Gly Asp Cys Asp Gln Phe Cys His Glu GluGln 130 135 140 AAC TCT GTG GTG TGC TCC TGC GCC CGC GGG TAC ACC CTG GCTGAC AAC 480 Asn Ser Val Val Cys Ser Cys Ala Arg Gly Tyr Thr Leu Ala AspAsn 145 150 155 160 GGC AAG GCC TGC ATT CCC ACA GGG CCC TAC CCC TGT GGGAAA CAG ACC 528 Gly Lys Ala Cys Ile Pro Thr Gly Pro Tyr Pro Cys Gly LysGln Thr 165 170 175 CTG GAA CGC AGG AAG AGG TCA GTG GCC CAG GCC ACC AGCAGC AGC GGG 576 Leu Glu Arg Arg Lys Arg Ser Val Ala Gln Ala Thr Ser SerSer Gly 180 185 190 GAG GCC CCT GAC AGC ATC ACA TGG AAG CCA TAT GAT GCAGCC GAC CTG 624 Glu Ala Pro Asp Ser Ile Thr Trp Lys Pro Tyr Asp Ala AlaAsp Leu 195 200 205 GAC CCC ACC GAG AAC CCC TTC GAC CTG CTT GAC TTC AACCAG ACG CAG 672 Asp Pro Thr Glu Asn Pro Phe Asp Leu Leu Asp Phe Asn GlnThr Gln 210 215 220 CCT GAG AGG GGC GAC AAC AAC CTC ACC AGG ATC GTG GGAGGC CAG GAA 720 Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg Ile Val Gly GlyGln Glu 225 230 235 240 TGC AAG GAC GGG GAG TGT CCC TGG CAG GCC CTG CTCATC AAT GAG GAA 768 Cys Lys Asp Gly Glu Cys Pro Trp Gln Ala Leu Leu IleAsn Glu Glu 245 250 255 AAC GAG GGT TTC TGT GGT GGA ACT ATT CTG AGC GAGTTC TAC ATC CTA 816 Asn Glu Gly Phe Cys Gly Gly Thr Ile Leu Ser Glu PheTyr Ile Leu 260 265 270 ACG GCA GCC CAC TGT CTC TAC CAA GCC AAG AGA TTCAAG GTG AGG GTA 864 Thr Ala Ala His Cys Leu Tyr Gln Ala Lys Arg Phe LysVal Arg Val 275 280 285 GGG GAC CGG AAC ACG GAG CAG GAG GAG GGC GGT GAGGCG GTG CAC GAG 912 Gly Asp Arg Asn Thr Glu Gln Glu Glu Gly Gly Glu AlaVal His Glu 290 295 300 GTG GAG GTG GTC ATC AAG CAC AAC CGG TTC ACA AAGGAG ACC TAT GAC 960 Val Glu Val Val Ile Lys His Asn Arg Phe Thr Lys GluThr Tyr Asp 305 310 315 320 TTC GAC ATC GCC GTG CTC CGG CTC AAG ACC CCCATC ACC TTC CGC ATG 1008 Phe Asp Ile Ala Val Leu Arg Leu Lys Thr Pro IleThr Phe Arg Met 325 330 335 AAC GTG GCG CCT GCC TGC CTC CCC GAG CGT GACTGG GCC GAG TCC ACG 1056 Asn Val Ala Pro Ala Cys Leu Pro Glu Arg Asp TrpAla Glu Ser Thr 340 345 350 CTG ATG ACG CAG AAG ACG GGG ATT GTG AGC GGCTTC GGG CGC ACC CAC 1104 Leu Met Thr Gln Lys Thr Gly Ile Val Ser Gly PheGly Arg Thr His 355 360 365 GAG AAG GGC CGG CAG TCC ACC AGG CTC AAG ATGCTG GAG GTG CCC TAC 1152 Glu Lys Gly Arg Gln Ser Thr Arg Leu Lys Met LeuGlu Val Pro Tyr 370 375 380 GTG GAC CGC AAC AGC TGC AAG CTG TCC AGC AGCTTC ATC ATC ACC CAG 1200 Val Asp Arg Asn Ser Cys Lys Leu Ser Ser Ser PheIle Ile Thr Gln 385 390 395 400 AAC ATG TTC TGT GCC GGC TAC GAC ACC AAGCAG GAG GAT GCC TGC CAG 1248 Asn Met Phe Cys Ala Gly Tyr Asp Thr Lys GlnGlu Asp Ala Cys Gln 405 410 415 GGG GAC AGC GGG GGC CCG CAC GTC ACC CGCTTC AAG GAC ACC TAC TTC 1296 Gly Asp Ser Gly Gly Pro His Val Thr Arg PheLys Asp Thr Tyr Phe 420 425 430 GTG ACA GGC ATC GTC AGC TGG GGA GAG AGCTGT GCC CGT AAG GGG AAG 1344 Val Thr Gly Ile Val Ser Trp Gly Glu Ser CysAla Arg Lys Gly Lys 435 440 445 TAC GGG ATC TAC ACC AAG GTC ACC GCC TTCCTC AAG TGG ATC GAC AGG 1392 Tyr Gly Ile Tyr Thr Lys Val Thr Ala Phe LeuLys Trp Ile Asp Arg 450 455 460 TCC ATG AAA ACC AGG GGC TTG CCC AAG GCCAAG AGC CAT GCC CCG GAG 1440 Ser Met Lys Thr Arg Gly Leu Pro Lys Ala LysSer His Ala Pro Glu 465 470 475 480 GTC ATA ACG TCC TCT CCA TTA AAG TGA1467 Val Ile Thr Ser Ser Pro Leu Lys 485 488 amino acids amino acidsingle linear protein 44 Met Gly Arg Pro Leu His Leu Val Leu Leu Ser AlaSer Leu Ala Gly 1 5 10 15 Leu Leu Leu Leu Gly Glu Ser Leu Phe Ile ArgArg Glu Gln Ala Asn 20 25 30 Asn Ile Leu Ala Arg Val Thr Arg Ala Asn SerPhe Leu Glu Glu Met 35 40 45 Lys Lys Gly His Leu Glu Arg Glu Cys Met GluGlu Thr Cys Ser Tyr 50 55 60 Glu Glu Ala Arg Glu Val Phe Glu Asp Ser AspLys Thr Asn Glu Phe 65 70 75 80 Trp Asn Lys Tyr Lys Asp Gly Asp Gln CysGlu Thr Ser Pro Cys Gln 85 90 95 Asn Gln Gly Lys Cys Lys Asp Gly Leu GlyGlu Tyr Thr Cys Thr Cys 100 105 110 Leu Glu Gly Phe Glu Gly Lys Asn CysGlu Leu Phe Thr Arg Lys Leu 115 120 125 Cys Ser Leu Asp Asn Gly Asp CysAsp Gln Phe Cys His Glu Glu Gln 130 135 140 Asn Ser Val Val Cys Ser CysAla Arg Gly Tyr Thr Leu Ala Asp Asn 145 150 155 160 Gly Lys Ala Cys IlePro Thr Gly Pro Tyr Pro Cys Gly Lys Gln Thr 165 170 175 Leu Glu Arg ArgLys Arg Ser Val Ala Gln Ala Thr Ser Ser Ser Gly 180 185 190 Glu Ala ProAsp Ser Ile Thr Trp Lys Pro Tyr Asp Ala Ala Asp Leu 195 200 205 Asp ProThr Glu Asn Pro Phe Asp Leu Leu Asp Phe Asn Gln Thr Gln 210 215 220 ProGlu Arg Gly Asp Asn Asn Leu Thr Arg Ile Val Gly Gly Gln Glu 225 230 235240 Cys Lys Asp Gly Glu Cys Pro Trp Gln Ala Leu Leu Ile Asn Glu Glu 245250 255 Asn Glu Gly Phe Cys Gly Gly Thr Ile Leu Ser Glu Phe Tyr Ile Leu260 265 270 Thr Ala Ala His Cys Leu Tyr Gln Ala Lys Arg Phe Lys Val ArgVal 275 280 285 Gly Asp Arg Asn Thr Glu Gln Glu Glu Gly Gly Glu Ala ValHis Glu 290 295 300 Val Glu Val Val Ile Lys His Asn Arg Phe Thr Lys GluThr Tyr Asp 305 310 315 320 Phe Asp Ile Ala Val Leu Arg Leu Lys Thr ProIle Thr Phe Arg Met 325 330 335 Asn Val Ala Pro Ala Cys Leu Pro Glu ArgAsp Trp Ala Glu Ser Thr 340 345 350 Leu Met Thr Gln Lys Thr Gly Ile ValSer Gly Phe Gly Arg Thr His 355 360 365 Glu Lys Gly Arg Gln Ser Thr ArgLeu Lys Met Leu Glu Val Pro Tyr 370 375 380 Val Asp Arg Asn Ser Cys LysLeu Ser Ser Ser Phe Ile Ile Thr Gln 385 390 395 400 Asn Met Phe Cys AlaGly Tyr Asp Thr Lys Gln Glu Asp Ala Cys Gln 405 410 415 Gly Asp Ser GlyGly Pro His Val Thr Arg Phe Lys Asp Thr Tyr Phe 420 425 430 Val Thr GlyIle Val Ser Trp Gly Glu Ser Cys Ala Arg Lys Gly Lys 435 440 445 Tyr GlyIle Tyr Thr Lys Val Thr Ala Phe Leu Lys Trp Ile Asp Arg 450 455 460 SerMet Lys Thr Arg Gly Leu Pro Lys Ala Lys Ser His Ala Pro Glu 465 470 475480 Val Ile Thr Ser Ser Pro Leu Lys 485 8 amino acids amino acid singlelinear peptide Modified-site 2...2 Xaa = Arg, Asp, Phe, Thr, Leu or Ser45 Gly Xaa Xaa Xaa Xaa Xaa Arg Xaa 1 5 8 amino acids amino acid singlelinear peptide 46 Gly Asp Asn Asn Leu Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 47 Gly Asp Gln Asn Leu Thr Arg Ile 1 58 amino acids amino acid single linear peptide 48 Gly Lys Asn Asn LeuThr Arg Ile 1 5 8 amino acids amino acid single linear peptide 49 GlyLys Gln Asn Leu Thr Arg Ile 1 5 8 amino acids amino acid single linearpeptide 50 Gly Phe Asn Asp Phe Thr Arg Val 1 5 8 amino acids amino acidsingle linear peptide 51 Gly Phe Gln Asp Phe Thr Arg Val 1 5 8 aminoacids amino acid single linear peptide 52 Gly Lys Asn Asp Phe Thr ArgVal 1 5 8 amino acids amino acid single linear peptide 53 Gly Lys GlnAsp Phe Thr Arg Val 1 5 8 amino acids amino acid single linear peptide54 Gly Phe Asn Asp Phe Thr Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 55 Gly Phe Gln Asp Phe Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 56 Gly Lys Asn Asp Phe Thr Arg Ile 1 58 amino acids amino acid single linear peptide 57 Gly Lys Gln Asp PheThr Arg Ile 1 5 8 amino acids amino acid single linear peptideModified-site 8...8 Xaa = Ile or Val 58 Gly Thr Lys Ile Lys Pro Arg Xaa1 5 8 amino acids amino acid single linear peptide Modified-site 8...8Xaa = Ile or Val 59 Gly Thr Gln Ile Lys Pro Arg Xaa 1 5 8 amino acidsamino acid single linear peptide Modified-site 8...8 Xaa = Ile or Val 60Gly Lys Lys Ile Lys Pro Arg Xaa 1 5 8 amino acids amino acid singlelinear peptide Modified-site 8...8 Xaa = Ile or Val 61 Gly Lys Gln IleLys Pro Arg Xaa 1 5 8 amino acids amino acid single linear peptide 62Gly Thr Lys Thr Ser Thr Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 63 Gly Thr Gln Thr Ser Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 64 Gly Lys Lys Thr Ser Thr Arg Ile 1 58 amino acids amino acid single linear peptide 65 Gly Lys Gln Thr SerThr Arg Ile 1 5 8 amino acids amino acid single linear peptideModified-site 8...8 Xaa = Ile or Val 66 Gly Leu Ser Ser Met Thr Arg Xaa1 5 8 amino acids amino acid single linear peptide Modified-site 8...8Xaa = Ile or Val 67 Gly Leu Gln Ser Met Thr Arg Xaa 1 5 8 amino acidsamino acid single linear peptide Modified-site 8...8 Xaa = Ile or Val 68Gly Lys Ser Ser Met Thr Arg Xaa 1 5 8 amino acids amino acid singlelinear peptide Modified-site 8...8 Xaa = Ile or Val 69 Gly Lys Gln SerMet Thr Arg Xaa 1 5 8 amino acids amino acid single linear peptide 70Gly Ser Lys Pro Gln Gly Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 71 Gly Ser Gln Pro Gln Gly Arg Ile 1 5 8 amino acidsamino acid single linear peptide 72 Gly Lys Lys Pro Gln Gly Arg Ile 1 58 amino acids amino acid single linear peptide 73 Gly Lys Gln Pro GlnGly Arg Ile 1 5 8 amino acids amino acid single linear peptide 74 GlyLys Gln Ile Glu Gly Arg Ile 1 5 8 amino acids amino acid single linearpeptide 75 Gly Lys Gln Met Lys Thr Arg Ile 1 5 8 amino acids amino acidsingle linear peptide 76 Gly Leu Glu Arg Arg Lys Arg Ile 1 5 8 aminoacids amino acid single linear peptide 77 Gly Leu Gln Arg Arg Lys ArgIle 1 5 8 amino acids amino acid single linear peptide 78 Gly Lys GluArg Arg Lys Arg Ile 1 5 8 amino acids amino acid single linear peptide79 Gly Lys Gln Arg Arg Lys Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 80 Gly Leu Ala Arg Val Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 81 Gly Leu Gln Arg Val Thr Arg Ile 1 58 amino acids amino acid single linear peptide 82 Gly Lys Ala Arg ValThr Arg Ile 1 5 8 amino acids amino acid single linear peptide 83 GlyLys Gln Arg Val Thr Arg Ile 1 5 8 amino acids amino acid single linearpeptide 84 Gly Leu Gln Arg Val Arg Arg Ile 1 5 8 amino acids amino acidsingle linear peptide 85 Gly Lys Gln Arg Val Arg Arg Ile 1 5 8 aminoacids amino acid single linear peptide 86 Gly Leu His Arg Arg Arg ArgIle 1 5 8 amino acids amino acid single linear peptide 87 Gly Leu GlnArg Arg Arg Arg Ile 1 5 8 amino acids amino acid single linear peptide88 Gly Lys His Arg Arg Arg Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 89 Gly Lys Gln Arg Arg Arg Arg Ile 1 5 8 amino acidsamino acid single linear peptide 90 Gly Leu Asn Arg Pro Lys Arg Ile 1 58 amino acids amino acid single linear peptide 91 Gly Leu Gln Arg ProLys Arg Ile 1 5 8 amino acids amino acid single linear peptide 92 GlyLys Asn Arg Pro Lys Arg Ile 1 5 8 amino acids amino acid single linearpeptide 93 Gly Lys Gln Arg Pro Lys Arg Ile 1 5 8 amino acids amino acidsingle linear peptide 94 Gly Leu Arg Ile Arg Lys Arg Ile 1 5 8 aminoacids amino acid single linear peptide 95 Gly Leu Gln Ile Arg Lys ArgIle 1 5 8 amino acids amino acid single linear peptide 96 Gly Lys ArgIle Arg Lys Arg Ile 1 5 8 amino acids amino acid single linear peptide97 Gly Lys Gln Ile Arg Lys Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 98 Gly Lys Gln Arg Ser Lys Arg Ile 1 5 8 amino acidsamino acid single linear peptide 99 Gly Lys Gln Arg Val Thr Arg Ile 1 58 amino acids amino acid single linear peptide 100 Gly Lys Gln Arg LeuLys Arg Ile 1 5 5 amino acids amino acid single linear peptide 101 ArgVal Thr Arg Ile 1 5 10 amino acids amino acid single linear peptide 102Thr Lys Glu Arg Arg Lys Arg Ser Val Ala 1 5 10 8 amino acids amino acidsingle linear peptide 103 Asn Leu Thr Arg Ile Val Gly Gly 1 5 8 aminoacids amino acid single linear peptide 104 Asp Phe Thr Arg Val Val GlyGly 1 5 8 amino acids amino acid single linear peptide 105 Thr Leu GluArg Thr Val Gly Gly 1 5 8 amino acids amino acid single linear peptide106 Ile Lys Pro Arg Ile Val Gly Gly 1 5 8 amino acids amino acid singlelinear peptide 107 Ser Met Thr Arg Ile Val Gly Gly 1 5 8 amino acidsamino acid single linear peptide 108 Met Lys Thr Arg Ile Val Gly Gly 1 58 amino acids amino acid single linear peptide 109 Ile Glu Gly Arg IleVal Gly Gly 1 5 8 amino acids amino acid single linear peptide 110 ArgArg Lys Arg Ile Val Gly Gly 1 5 8 amino acids amino acid single linearpeptide 111 Arg Val Arg Arg Ile Val Gly Gly 1 5 8 amino acids amino acidsingle linear peptide 112 Arg Arg Arg Arg Ile Val Gly Gly 1 5 8 aminoacids amino acid single linear peptide 113 Arg Pro Lys Arg Ile Val GlyGly 1 5 8 amino acids amino acid single linear peptide 114 Ile Arg LysArg Ile Val Gly Gly 1 5 8 amino acids amino acid single linear peptide115 Arg Ser Lys Arg Ile Val Gly Gly 1 5 8 amino acids amino acid singlelinear peptide 116 Arg Val Thr Arg Ile Val Gly Gly 1 5 8 amino acidsamino acid single linear peptide 117 Arg Leu Lys Arg Ile Val Gly Gly 1 58 amino acids amino acid single linear peptide 118 Pro Gln Gly Arg IleVal Gly Gly 1 5 8 amino acids amino acid single linear peptide 119 ThrSer Thr Arg Ile Val Gly Gly 1 5 4 amino acids amino acid single linearpeptide 120 Met Lys Thr Arg 1 8 amino acids amino acid single linearpeptide 121 Xaa Xaa Xaa Arg Xaa Val Gly Gly 1 5 5 amino acids amino acidsingle linear peptide 122 Met Lys Thr Lys Gly 1 5 10 amino acids aminoacid single linear peptide 123 Gly Glu Ser Leu Phe Ile Arg Arg Glu Gln 15 10 14 amino acids amino acid single linear peptide 124 Ile Leu Ala ArgVal Thr Arg Ala Asn Ser Phe Leu Glu Glu 1 5 10 7 amino acids amino acidsingle linear peptide 125 Ser Val Ala Gln Ala Thr Ser 1 5 7 amino acidsamino acid single linear peptide 126 Leu Phe Ile Arg Arg Glu Gln 1 5 7amino acids amino acid single linear peptide 127 Ala Asn Ser Phe Leu GluGlu 1 5 10 amino acids amino acid single linear peptide 128 Val Thr ArgAla Asn Ser Phe Leu Glu Glu 1 5 10 5 amino acids amino acid singlelinear peptide 129 Arg Val Thr Arg Ala 1 5 5 amino acids amino acidsingle linear peptide 130 Arg Arg Lys Arg Ser 1 5 5 amino acids aminoacid single linear peptide Modified-site 3...3 Xaa = Lys or Arg 131 ArgXaa Xaa Arg Xaa 1 5 5 amino acids amino acid single linear peptide 132Asp Phe Thr Arg Val 1 5 5 amino acids amino acid single linear peptide133 Ile Lys Pro Arg Ile 1 5 5 amino acids amino acid single linearpeptide 134 Ser Met Thr Arg Ile 1 5 5 amino acids amino acid singlelinear peptide 135 Met Lys Thr Arg Ile 1 5 5 amino acids amino acidsingle linear peptide 136 Ile Glu Gly Arg Ile 1 5 5 amino acids aminoacid single linear peptide 137 Arg Arg Lys Arg Ile 1 5 5 amino acidsamino acid single linear peptide 138 Arg Val Arg Arg Ile 1 5 5 aminoacids amino acid single linear peptide 139 Arg Arg Arg Arg Ile 1 5 5amino acids amino acid single linear peptide 140 Arg Pro Lys Arg Ile 1 55 amino acids amino acid single linear peptide 141 Ile Arg Lys Arg Ile 15 5 amino acids amino acid single linear peptide 142 Arg Ser Lys Arg Ile1 5 5 amino acids amino acid single linear peptide 143 Arg Leu Lys ArgIle 1 5 5 amino acids amino acid single linear peptide 144 Pro Gln GlyArg Ile 1 5 5 amino acids amino acid single linear peptide 145 Thr SerThr Arg Ile 1 5

What is claimed is:
 1. A factor XΔ analogue comprising a factor X aminoacid sequence in which amino acids Arg180 to Arg234 of SEQ ID NO:44 aredeleted, and having a modification in said amino acid sequence betweenGly173 and Arg179 of SEQ ID NO:44, said modification resulting in aprocessing site for a protease that does not naturally cleave betweenGly173 and Arg179 of SEQ ID NO:44.
 2. A factor XΔ analogue as set forthin claim 1, wherein said modification is at least one amino acidexchange in the region of said amino acid sequence between Gly173 andArg179 of SEQ ID NO:44.
 3. A factor XΔ analogue as set forth in claim 1,comprising a factor X sequence wherein amino acids Gly173 to Arg179 andresidue 235 of SEQ ID NO:44 have the sequenceGly173-R6-R5-R4-R3-R2-Arg179/R1(235), wherein a) R1 is an amino acidselected from the group consisting of Val, Ser, Thr, Ile and Ala, b) R2is an amino acid selected from the group consisting of Glu, Thr, Pro,Gly, Lys and Arg, c) R3 is an amino acid selected from the groupconsisting of Leu, Phe, Lys, Met, Gln, Glu, Ser, Val, Arg and Pro, d) R4is an amino acid selected from the group consisting of Thr, Asp, Asn,Ile, Ser, Met, Pro, Arg and Lys, e) R5 is an amino acid selected fromthe group consisting of Asn, Lys, Ser, Glu, Gin, Ala, His and Arg, andf) R6 is an amino acid selected from the group consisting of Asp, Phe,Thr, Arg, Leu and Ser.
 4. A factor XΔ analogue as set forth in claim 1,wherein the protease is selected from the group consisting of anendoprotease, a serine protease and a derivative of these proteases. 5.A factor XΔ analogue as set forth in claim 4, wherein said endoproteaseis selected from the group consisting of kexin/Kex2, furin/PACE,PC1/PC3, PC2, PC4, PACE 4 and LPC/PC7 and said serine protease isselected from the group consisting of factor IIa, factor VIIa, factorIXa, factor XIIa, factor XIa, factor Xa and kallikrein.
 6. A factor XΔanalogue as set forth in claim 1, wherein said factor XΔ analogue ispresent as a single chain polypeptide in enzymatically inactive form. 7.A factor XΔ analogue as set forth in claim 6, wherein said modificationpermits activation of the inactive, single-chain factor XΔ analoguepolypeptide into the double-chain, active factor Xa analogue form.
 8. Afactor XΔ analogue as set forth in claim 1, further comprising a furthermodification at Lys370 and/or within a segment extending from Arg469 toLys488 of SEQ ID NO:44.
 9. A factor XΔ analogue as set forth in claim 8,wherein said further modification is located at a β-peptide cleavagesite located between Arg469 and Gly470 of SEQ ID NO:44.
 10. A factor XΔanalogue as set forth in claim 8, wherein said further modification isselected from the group consisting of a mutation, a deletion and aninsertion, and is located between amino acid positions Arg469 and Ser476of SEQ ID NO:44.
 11. A factor XΔ analogue as set forth in claim 8,wherein said further modification prevents β-peptide from being cleavedoff, the β-peptide extending from Gly470 to Lys488 of SEQ ID NO:44. 12.A factor XΔ analogue as set forth in claim 8, wherein said furthermodification is a deletion of the factor X β-peptide, which peptideextends from Gly470 to Lys488 of SEQ ID NO:44.
 13. A factor XΔ analogueas set forth in claim 8, wherein the further modification is atranslation stop signal.
 14. A factor XΔ analogue as set forth in claim13, wherein said translation stop signal is at the position of aminoacid Gly470 of the factor X amino acid sequence.
 15. A factor XΔanalogue as set forth in claim 6, wherein said modification in saidamino acid sequence between Gly173 and Arg179 permits an in vitroactivation of the inactive factor XΔ analogue to active factor XΔanalogue.
 16. A factor XΔ analogue as set forth in claim 15, whereinsaid in vitro activation is effected by a protease selected from thegroup consisting of an endoprotease, a serine protease, and a derivativeof these proteases.
 17. A factor XΔ analogue as set forth in claim 16,wherein said endoprotease is selected from the group consisting ofkexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7, and saidserine protease is selected from the group consisting of factor IIa,factor VIIa, factor IXa, factor XIIa, factor XIa, factor Xa andkallikrein.
 18. A factor XΔ analogue as set forth in claim 1, comprisingan intact β-peptide, wherein the β-peptide extends from Gly470 to Lys488of SEQ ID NO:44, and the factor XΔ analogue is factor XΔα.
 19. Apreparation comprising purified factor XΔ analogue, said factor XΔanalogue having a factor X amino acid sequence in which amino acidsArg180 to Arg234 of SEQ ID NO:44 are deleted, and having a modificationin said amino acid sequence between Gly173 and Arg179 of SEQ ID NO:44,said modification resulting in a processing site for a protease thatdoes not naturally cleave between Gly173 and Arg179 of SEQ ID NO:44. 20.A preparation as set forth in claim 19, wherein said purified factor XΔanalogue is a single-chain factor XΔ analogue in enzymatically inactiveform and having a purity of at least 80%, said purified factor XΔanalogue being free from inactive, proteolytic intermediates formedduring processing or autoproteolysis of factor XΔ analogue or factor Xaanalogue.
 21. A preparation as set forth in claim 20, wherein saidpurity of said factor XΔ analogue is at least 90%.
 22. A preparation asset forth in claim 20, wherein said purity of said factor XΔ analogue isat least 95%.
 23. A preparation as set forth in claim 19, wherein thefactor XΔ analogue is factor XΔα.
 24. A preparation as set forth inclaim 19, wherein the factor XΔ analogue is factor XΔβ.
 25. Apreparation as set forth in claim 19, wherein said factor XΔ analogue isa single-chain molecule in isolated form.
 26. A preparation as set forthin claim 19, wherein the factor XΔ analogue of the preparation retains100% of its original activity for at least a day.
 27. A preparation asset forth in claim 25, wherein said modification permits in vitroactivation of single-chain factor XΔ analogue to active factor Xaanalogue.
 28. A preparation as set forth in claim 19, said preparationbeing formulated as a pharmaceutical preparation.
 29. A preparation asset forth in claim 28, said preparation being contained in a device incombination with a protease selected from the group consisting of anendoprotease, a serine protease and a derivative thereof.
 30. Apreparation as set forth in claim 29, wherein said endoprotease isselected from the group consisting of kexin/Kex2, furin/PACE, PC1/PC3,PC2, PC4, PACE 4, and LPC/PC7, and said serine protease is selected fromthe group consisting of factor IIa, factor VIIa, factor IXa, factorXIIa, factor XIa, factor Xa and kallikrein.
 31. A preparation as setforth in claim 29, wherein said device is an application device adaptedfor administration of the preparation to a patient.
 32. A preparation asset forth in claim 28, wherein said preparation and said protease arepresent separately in said device.
 33. A preparation comprising apurified factor Xa analogue free from inactive intermediates formedduring processing or autoproteolysis of factor XΔ analogue or factor Xaanalogue, obtainable by activation of a factor XΔ analogue comprising afactor X amino acid sequence in which amino acids Arg180 to Arg234 ofSEQ ID NO:44 are deleted, and having a modification in said amino acidsequence between Gly173 and Arg179 of SEQ ID NO:44, said modificationresulting in a processing site for a protease that does not naturallycleave between Gly173 and Arg179 of SEQ ID NO:44.
 34. A preparation asset forth in claim 33, wherein the active factor Xa analogue is adouble-chain molecule in isolated form.
 35. A preparation as set forthin claim 33, wherein said factor Xa analogue has a purity of at least80%.
 36. A preparation as set forth in claim 35, wherein said purity ofsaid factor Xa analogue is at least 90%.
 37. A preparation as set forthin claim 35, wherein said purity of said factor Xa analogue is at least95%.
 38. A preparation as set forth in claim 33, further comprising aphysiologically acceptable carrier and being present in storage-stableform.
 39. A preparation as set forth in claim 38, further comprising acomponent selected from the group consisting of a blood factor and anactivated form of a blood factor.
 40. A preparation as set forth inclaim 39, wherein said component is at least one component having factorVIII bypass activity.
 41. A preparation as set forth in claim 33, saidpreparation being formulated as a pharmaceutical composition.
 42. Amethod for treating and preventing blood coagulation disorders inpatients, comprising administering an effective dose of a preparationcomprising purified factor XΔ analogue, said factor XΔ analoguecomprising a factor X amino acid sequence in which amino acids Arg180 toArg234 of SEQ ID NO:44 are deleted, and having a modification in saidamino acid sequence between Gly173 and Arg179 of SEQ ID NO:44, saidmodification resulting in a processing site for a protease that does notnaturally cleave between Gly173 and Arg179 of SEQ ID NO:44.
 43. A methodas set forth in claim 42, wherein said blood coagulation disorder ishemophilia.
 44. A method as set forth in claim 42, wherein said bloodcoagulation disorder involves inhibitor antibody formation inhemophiliacs.
 45. A method of preparing a preparation comprisingpurified recombinant factor XΔ analogue, said method comprising. a)providing a preparation comprising a recombinant factor XΔ analogue,wherein said factor XΔ analogue has a factor X amino acid sequence inwhich amino acid Arg180 to Arg234 of SEQ ID NO:44 are deleted, andhaving a modification in said amino acid sequence between Gly173 andArg179 of SEQ ID NO:44, said modification resulting in a processing sitefor a protease that does not naturally cleave between Gly173 and Arg179of SEQ ID NO:44; b) isolating said recombinant factor XΔ analogue as asingle-chain polypeptide; and c) purifying said isolated, single-chainfactor, recombinant XΔ analogue polypeptide.
 46. A method as set forthin claim 45, wherein said providing step (a) comprises providing anucleic acid encoding said factor XΔ analogue; transfecting a suitablecell; and expressing said factor XΔ analogue.
 47. A method of preparinga preparation comprising factor Xa analogue, said method comprising: a)providing a preparation comprising a recombinant preparation of factorXΔ analogue, wherein said factor XΔ analogue has a factor X amino-acidsequence in which amino acid Arg180 to Arg234 of SEQ ID NO:44 aredeleted, and having a modification in said amino acid sequence betweenGly173 and Arg179 of SEQ ID NO:44, said modification resulting in aprocessing site for a protease that does not naturally cleave betweenGly173 and Arg179 of SEQ ID NO:44; b) isolating said recombinant factorXΔ analogue as a single-chain polypeptide; c) purifying said isolated,single-chain, recombinant factor XΔ analogue polypeptide; and d)subjecting said purified single-chain, recombinant factor XΔ analoguepolypeptide to an activation step to obtain said preparation containingactivated factor Xa analogue.
 48. A method as set forth in claim 47,wherein the activation step comprises contacting said single-chain,recombinant factor XΔ analogue polypeptide with a protease selected fromthe group consisting of an endoprotease, a serine protease and aderivative of these proteases, under conditions permitting cleavage ofsaid single-chain, recombinant factor XΔ analogue polypeptide into thedouble-chain factor Xa analogue form.
 49. A method as set forth in claim48, wherein said endoprotease is selected from the group consisting ofkexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE 4 and LPC/PC7, and saidserine protease is selected from the group consisting of factor IIa,factor VIIa, factor IXa, factor XIIa, factor XIa, factor Xa andkallikrein.
 50. A method as set forth in claim 48, wherein said proteaseis immobilized.
 51. A method as set forth in claim 47, wherein saidfactor Xa analogue is free from inactive intermediates formed duringprocessing or autoproteolysis of factor XΔ analogue or factor Xaanalogue.
 52. A factor XΔ analogue as set forth in claim 8, wherein saidfurther modification is located between amino acid positions Arg469 andSer476, and/or at Lys370 of SEQ ID NO:44.
 53. A factor XΔ analogue asset forth in claim 13, wherein said translation stop signal is atresidues Lys370, Gly470, Ala474, or Ser476 of SEQ ID NO:44.
 54. A factorXΔ analogue as set forth in claim 8, wherein said further modificationis an amino acid substitution at one or more of the amino acid positionsArg469, Gly470, Lys370, Lys475 and Ser476 of said factor X amino acidsequence.
 55. A preparation as set forth in claim 28, further comprisinga component selected from the group consisting of a blood factor and anactivated form of a blood factor.
 56. A preparation as set forth inclaim 55, wherein said component is at least one component having factorVIII bypass activity.